Immunomodulatory Compositions, Methods and Systems Comprising Immunogenic Fragments of ApoB-100

ABSTRACT

Immunostimulatory agents, T cell, compositions, methods and systems for treating and/or preventing various conditions in a human individual.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application Ser. No. 61/413,378 entitled “Immunomodulatory Compositions, Methods and Systems Comprising Immunogenic Fragments of Apob100” filed on Nov. 12, 2010, with docket number P700-USP, which is herein incorporated by reference in its entirety. The present application is also related to PCT application WO 02/080954 filed on Apr. 5, 2002, to PCT application Ser. No. ______ entitled “Immunomodulatory Methods and Systems for Treatment and/or Prevention of Aneurysms” filed on Nov. 11, 2011 with docket number P686-PCT, and to PCT application Ser. No. ______ entitled “Immunomodulatory Methods and Systems for Treatment and/or Prevention of Hypertension” filed on Nov. 11, 2011 with attorney docket P694-PCT, each of which is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates to immunomodulatory compositions methods, systems, compositions, and in particular vaccines that comprise immunogenic fragments of ApoB100 are suitable for the treatment or prevention of conditions such as atherosclerosis, aneurysms, hypertension and/or of a condition associated thereto.

BACKGROUND

Immunogenic fragments of ApoB100 have been associated with treatment and/or prevention to various conditions in an individual.

In particular, certain immunogenic fragments of ApoB100 have been associated with treatment of atherosclerosis in individuals.

SUMMARY

Provided herein are compositions, methods and systems that allow in several embodiments treatment and/or prevention of various conditions treated with immunogenic fragments of ApoB100 in a human individual.

According to a first aspect, a method to treat and/or prevent a condition in a human individual, is described the condition treatable and/or preventable by administering one or more immunogenic fragments of ApoB100 or an immunogenically active portion thereof. The method comprises administering to an individual the one or more immunogenic fragment of apoB-100 or the immunogenically active portion thereof at a concentration of less than 1 mg.

According to a second aspect, a pharmaceutical composition comprising less than 1 mg of one or more immunogenic fragments of ApoB100 or an immunogenically active portion thereof and a pharmaceutically acceptable vehicle.

The compositions, methods and systems herein described can be used in connection with applications wherein treatment of a condition treatable and/or preventable by administering one or more immunogenic fragments of ApoB100 or an immunogenically active portion thereof in a human individual is desired.

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the examples, serve to explain the principles and implementations of the disclosure.

FIG. 1 shows a representation of the locations of the segments examined for average diameter of segmental aneurysm according to an embodiment herein described.

FIG. 2 shows a Kaplan Meier survival curve for mice immunized with or without p210 according to an embodiment herein described.

FIG. 3 shows p210 immunization confers athero-protective effect. (A) Immunization with native p210 resulted in a significant reduction in aortic atherosclerosis when compared to PBS and cBSA/Alum group (n=9-10 each group, representative picture from each group shown). (B) P210 immunization significantly reduced macrophage infiltration and DC presence assessed by MOMA-2 (n=9-10 each group) and CD11c (n=7-12 each group) immuno-reactivity, respectively in aortic sinus plaques.

FIG. 4 Effect of p210 immunization on DCs. One week after primary immunization, (A) CD11c(+) or (B) CD11c(+)CD86(+) cells at the immunization sites was significantly reduced in p210/cBSA/alum group when compared to cBSA/alum group. N=10 each group. (C) One week after third immunization, p210 immunized mice had reduced CD11c(+)CD86(+) cells in lymph nodes compared to cBSA/alum group (n=5 in each group; ANOVA followed by multiple group comparison).

FIG. 5 shows IgM or IgG titer against p210 before and after p210 immunization. (A) The p210 IgG titers were low before immunization and remained low in the PBS group at euthanasia but significantly increased in cBSA/alum and p210/cBSA/alum groups, with the highest titer in the cBSA/alum group. (B) The p210 IgM titers were low before immunization and significantly increased at euthanasia with no difference among 3 groups of mice. N=5 for 6-7 week time-point and n=9 for 25 week time-point.

FIG. 6 shows activated lymphocyte population after immunization in vivo. (A) CD8(+)CD25(+) T-cell population in the lymph nodes was significantly higher in p210/cBSA/alum group when compared to that of PBS or cBSA/alum groups; (B) CD4(+)CD25(+) T-cells in the lymph nodes did not differ among the three groups. There was a significantly larger population of splenic CD8(+)CD25(+)IL-10(+) T-cells in p210/cBSA/alum group among 3 groups (C) without difference in splenic CD8(+)CD25(+)IL12(+) T-cells among 3 groups (D). Splenic CD4(+)CD25(+)IL-10(+) T-cell population significantly increased in the cBSA/alum group, but was significantly attenuated by the p210/cBSA/alum immunization (E) and (F) splenic CD4(+)CD25(+)IL12(+) T-cells did not differ among 3 groups. N=9-10 in each group for (A) and (B); n=5 in each group for (C), (D), (E) and (F).

FIG. 7 shows adoptive transfer of CD8(+) T-cells from p210 immunized donors recapitulated the athero-protective effect of p210 immunization but not by transfer of B-cells or CD4(+)CD25(+) T-cells. (A) The recipient mice of CD8(+) T-cells from p210/cBSA/alum immunized donors developed significantly smaller atherosclerotic lesions compared to the recipient mice of CD8(+) T-cells from other 2 groups (n=9-10 each group). (B) Adoptive transfer of B-cells from p210/cBSA/alum donors did not reduce atherosclerosis when compared to the recipient mice of B-cells from PBS or cBSA/alum groups (n=9 each group). Recipient mice of CD4(+)CD25(+) T-cells (n=9-13 each group) with 2 different doses (C. 1×10⁵ cells/mouse or D. 3×10⁵ cells/mouse) did not reproduce the athero-reducing effect of p210 immunization.

FIG. 8 shows increased cytolytic activity of CD8(+) T cells from p210 immunized mice against dendritic cells in vitro. CD8(+) T-cells from p210 immunized mice significantly had a higher cytolytic activity against dendritic cells when compared to those from PBS or BSA/alum groups. Experiments were repeated 4 times with CD8(+) T-cells pooled from 5 mice in each group each time. Duplicate or triplicate was done each time with total of 11 data-points in each group altogether.

FIG. 9 shows CD8(+) T-cells from p210 immunized mice containing higher level of Granzyme B when compared to those from PBS or cBSA/alum group; whereas there is no difference in perforin level.

FIG. 10 shows IgG titers against KLH or TNP after p210 immunization. (A) Prior immunization with p210 did not affect the efficacy of subsequent T-cell dependent (KLH, n=3-6 each group) or (B) T-cell independent (TNP, n=4-5 each group) immunization as assessed by the IgG antibody titers when compared to mice received PBS or cBSA/alum.

FIG. 11 shows the effect of p210 immunization on mean blood pressure in various groups of mice according to an embodiment herein described.

FIGS. 12A and 12B shows the effect of p210 immunization on heart rate and mean blood pressure in various groups of mice according to embodiments herein described.

FIG. 13 shows a Kaplan Meier survival curve for mice immunized with or without p210 according to one embodiment herein described.

FIG. 14 shows Antibody response to p210 in apoE−/− mice according one embodiment herein described.

FIG. 15 shows cytolytic activity of p210-immune CD8⁺ T cells is abrogated by depletion of CD25⁺ cells. Lytic activity specific to p210 is also abrogated by absence of serum lipids in the assay medium.

FIG. 16 shows endocytosis of FITC-labeled p210 by DCs according one embodiment herein described.

FIG. 17 shows presentation of the peptide p210 by DCs to CD8⁺ CD25⁻ T cells in vitro as shown by increased activated CD25+ cells according one embodiment herein described.

FIG. 18 shows CD8⁺ lytic activity gated on FITC cells according an embodiment herein described. p210-specific lytic activity by CD8⁺ T cells from p210-vaccinated mice using DCs loaded with FITC-labeled p210.

DETAILED DESCRIPTION

Methods and systems are herein described that allow in several embodiments, treatment and/or prevention of various conditions in a human individual.

The term “treat,” or “treating” or “treatment” as used herein indicates any activity that is part of a medical care for, or that deals with, a condition medically or surgically. The term “preventing” or “prevention” as used herein indicates any activity, which reduces the burden of mortality or morbidity from a condition in an individual. This takes place at primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications.

The term “condition” as used herein indicates the physical status of the body of an individual (as a whole or of one or more of its parts) that does not conform to a physical status of the individual (as a whole or of one or more of its parts) that is associated with a state of complete physical, mental and possibly social well-being. Conditions herein described include but are not limited to disorders and diseases wherein the term “disorder” indicates a condition of the living individual that is associated to a functional abnormality of the body or of any of its parts, and the term “disease” indicates a condition of the living individual that impairs normal functioning of the body or of any of its parts and is typically manifested by distinguishing signs and symptoms. Exemplary conditions include but are not limited to injuries, disabilities, disorders (including mental and physical disorders), syndromes, infections, deviant behaviours of the individual and atypical variations of structure and functions of the body of an individual or parts thereof.

In some embodiments, treatment and/or prevention of various conditions can be provided by administering to a human individual an effective amount of one or more immunogenic fragments of ApoB100 or an immunogenically active portion thereof, wherein the effective amount is less than 1 mg.

The term “administer” or “administering” or “administration” as used herein means any method of providing an individual with a substance in any fashion including, but not limited to, those discussed herein.

The term “individual” or “individuals” as used herein indicates a single biological organism such as higher animals and in particular vertebrates such as mammals and more particularly human beings. In some embodiments, the individual has been previously identified as having an increased risk of aneurysm based on the detection of conditions typically associated with an increased risk of aneurysm (e.g. higher blood pressure, atherosclerosis). In some embodiments, the individual has not been identified as having an increased risk of aneurysm. In some embodiments, no investigation as to the risk for aneurysm in the individual has been performed.

The term “immunogenic fragment” or “antigenic fragment” as used herein indicates a portion of a polypeptide of any length capable of generating an immune response, such as an antigen. An antigen is a molecule recognized by the immune system. An antigenic fragment of ApoB100 is accordingly a portion of ApoB-100 that presents antigenic properties. The ability of a fragment or other molecule to generate an immune response and in particular a cellular and/or humoral response can be detected with techniques and procedures identifiable by a skilled person.

The term fragment in the sense of the present disclosure comprises not only fragments of any length from ApoB100, but also peptides produced by genetic recombination or chemically synthesized. The term “immunogenic fragments” in the sense of the present disclosure further comprise also derivative of any fragment, such as oxidative derivative and/or peptide treated with MDA or copper, which maintain a detectable antigenic property of the original fragment.

The term “derivative” as used herein with reference to a first peptide (e.g., an immunogenic fragment), indicates a second peptide that is structurally related to the first polypeptide and is derivable from the first polypeptide by a modification that introduces a feature that is not present in the first peptide while retaining functional properties of the first peptide. Accordingly, a derivative peptide of an immunogenic fragment, or of any portion thereof, e.g. an epitope thereof, usually differs from the original an immunogenic fragment or portion thereof by modification of the amino acidic sequence that might or might not be associated with an additional function not present in the original peptide or portion thereof. A derivative peptide of an immunogenic fragment or of any portion thereof retains however one or more of the immunogenic activities that are herein described in connection with an immunogenic fragment or portion thereof. The antigenic properties can be verified with methods and systems such as the ones already described for the immunogenic fragments and additional methods and systems identifiable to a skilled person. Typically, a derivative of an immunogenic fragment comprises at least one epitope of the immunogenic fragment.

The term immunogenically active portion in the sense of the present disclosure indicates any part of a reference antigen that can elicit specific immune response. Exemplary immunogenically active portions are the epitopes formed by 5 or more residues comprised within an immunogenic fragment. In some embodiments, epitopes within one fragment can overlap.

Immunogenic fragments can be expressed by recombinant technology, such as a fusion with an affinity or epitope tag, chemical synthesis of an oligopeptide, either free or conjugated to carrier proteins, or any other methods known in the art to express the ApoB-100 peptides.

Exemplary fragments of ApoB100 are peptides each comprising one of the sequences listed in the Sequence Listing as SEQ ID NO: 1 to SEQ ID NO: 302 described in further detail in the Examples section. Methods and systems suitable to identify an immunogenic fragment in the sense of the present are described in WO 02/080954, hereby incorporated by reference. Additional methods are exemplified in the Examples section (see e.g. Example 1). The term “protein” or “polypeptide” or “peptide” as used herein indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called an oligopeptide. As used herein the term “amino acid”, “amino acidic monomer”, or “amino acid residue” refers to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers. The term “amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, isotope, or with a different functional group but is otherwise identical to its natural amino acid analog. In some embodiments, the one or more immunogenic fragments of ApoB100 is associated to atherosclerosis reduction.

Methods to identify a molecule associated with atherosclerosis reduction are identifiable by a skilled person and include the exemplary procedures described in WO 02/080954 herein incorporated by reference in its entirety. In particular, the ability of a molecule to reduce atherosclerosis can be tested in an animal model following administration of the molecule in a suitable amount using procedure identifiable by a skilled person. For example following subcutaneous administration of a molecule herein described the ability of the molecule to affect atherosclerosis can be tested in mice as illustrated in the Examples sections. A skilled person will be able to identify additional procedure, schedule of administration and dosages upon reading of the present disclosure.

Accordingly in an exemplary embodiment, immunogenic molecule associated with atherosclerosis reduction can be identified by identifying a candidate immunogenic molecule able to provide a cellular and/or humoral response in the individual of interest; and testing the candidate immunogenic molecule for an ability to reduce atherosclerosis, to select the candidate immunogenic molecule associated with atherosclerosis reduction.

In particular, in some embodiments, immunogenic fragments of ApoB100 are immunogenic fragments producing an immune response associated to atherosclerosis reduction in the individual or in an animal model. In some of those embodiments, a percentage atherosclerosis reduction is at least about 20%, or at least about 30%, from about 40% to about 60% or about 50% to about 80%.

In some embodiments, the immunogenic fragment comprises at least one of peptide, each comprising p1 (SEQ ID NO: 1), p2 (SEQ ID NO: 2), p 11 (SEQ ID NO:11), p25 (SEQ ID NO:25), p45 (SEQ ID NO:45), p74 (SEQ ID NO:74), p99 (SEQ ID NO:99), p100 (SEQ ID NO:100), p102 (SEQ ID NO:102), p103 (SEQ ID NO: 103), p105 (SEQ ID NO:105), p129 (SEQ ID NO:129), p143 (SEQ ID NO:143), p148 (SEQ ID NO:148), p210 (SEQ ID NO:210), or p301 (SEQ ID NO:301).

In an embodiment, the one or more immunogenic fragments comprise one or more peptides each comprising p2 (SEQ ID NO:2), p 11 (SEQ ID NO:11), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), p148 (SEQ ID NO:148), or p210 (SEQ ID NO:210).

In an embodiment, the one or more immunogenic fragments comprise two peptides each comprising p143 (SEQ ID NO: 143), or p210 (SEQ ID NO:210). In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprises three peptides each comprising, one of p 11 (SEQ ID NO:11), p25 (SEQ ID NO: 25), or p74 (SEQ ID NO:74). In an embodiment, the one or more immunogenic fragments associated to atherosclerosis reduction comprises five peptides each comprising one of p99 (SEQ ID NO: 99), p100 (SEQ ID NO: 100), p102 (SEQ ID NO: 102), p103 (SEQ ID NO: 103), and p105 (SEQ ID NO: 105).

In an embodiment, the one or more immunogenic fragments comprise one or more peptides each comprising p2 (SEQ ID NO: 2), p45 (SEQ ID NO: 45), p74 (SEQ ID NO: 74), p102 (SEQ ID NO: 102), or p210 (SEQ ID NO:210).

In an embodiment, the one or more immunogenic fragments comprise a peptide comprising amino acids 16-35 of human apoB-100 (p2; SEQ ID NO:2).

In an embodiment the one or more immunogenic fragments comprise a peptide comprising amino acids 661-680 of human apoB-100 (p45; SEQ ID NO:45).

In an embodiment, the one or more immunogenic fragments comprise a peptide comprising amino acids 3136-3155 of human apoB-100 (P210; SEQ ID NO: 210).

In an embodiment, the one or more immunogenic fragments comprise a peptide comprising amino acids 4502-4521 of human apoB-100 (P301; SEQ ID NO: 301).

In an embodiment, the one or more immunogenic fragments comprise a peptide comprising amino acids 1-20 of human apoB-100 (P1; SEQ ID NO: 1).

Exemplary data showing association of the above peptides to atherosclerosis reduction are shown in Example 5 of the present disclosure and in International application WO 02/080954, herein incorporated by reference in its entirety (see in particular Table 1, Table 2, Table A and Table B). In particular for some of those peptides or combination thereof a percentage reduction of 64.6% (p143 and p210), 59.6% (p11, p25 and p174), 56.8% (p129, p148, and p167), p67.7 (p2), 57.9% (p210), 55.2% (p301), 47.4% (p45), 31% (p1) has been detected (see W0/02080954 incorporated herein by reference in its entirety, and in particular Table B).

Immunogenic peptides comprising any of the sequences herein described or immunogenically active portions of those peptides are identifiable by a skilled person using in silico and/or in vitro approaches. For example, in silico methods can be used to identify any of said epitopes or immunogenic peptides based on any of the sequences herein described. Reference is made for example, to the papers [44] to [51] each of which are incorporated herein by reference in its entirety.

Such papers describe various algorithms such as Tepitope (Radrizzani et al 2000), Adept (Maksuytov et al 1993), antigenic index (Jameson et al 1988) and others which can be used to identify the immunogenic molecules comprising the sequences at issue or any relevant epitopes.

Additional tests and laboratory procedures in vitro and/or in vivo suitable to be used alone or in connection with the identification in silico (e.g. ELISA, antigen-specific T cell proliferation assay, ELISPOT, antibody measurement) are identifiable by a skilled person that can be used by a skilled person to verify the in silico data and/or identify immunologically active molecules comprising any of the sequences herein described or immunologically active portions of those sequences.

Accordingly, in an exemplary embodiments, immunogenic peptides, herein described, immunogenically active portions thereof as well as derivative thereof can be identified by identifying candidate peptides, candidate active portion and/or candidate derivative by in silico analysis of any one of the sequences herein described, and by identifying the immunogenic peptides, immunogenically active portions and/or derivative by in vitro and/or in vivo testing of the candidate peptides, candidate active portion and/or candidate derivative. In particular, the in silico analysis can be performed by analyzing the sequence of the candidate with algorithm suitable to identify immunogenicity of a molecule or portion thereof. Similarly, the in vitro and/or in vivo testing comprises methods directed to identify immunogenicity of the candidate peptide, candidate active portion and/or derivative as well as effects of those molecules on aneurysm, with particular reference to formation or regression. Suitable methods and techniques are identifiable by a skilled person upon reading of the present disclosure.

In several embodiments, the immunogenic peptides, active portions thereof and derivative thereof are expected to include a sequence of at least about 5 amino acids, consistently with the typical length of epitopes as indicated in WO 02/080954 herein incorporated by reference in its entirety.

Additional conditions treatable with immunogenic fragments or an immunogenically active portion thereof herein described comprise aneurysmand hypertension.

The term “aneurysm” as used herein indicates a localized blood filled dilation of a blood vessel or of a portion thereof. In particular, an aneurysm can be an abnormal widening or ballooning of a portion of an artery due to weakness in the wall of the blood vessel, and can occur within any vasculature in the body. Aneurysms can be “true” in which the inner layers of a blood vessel bulges outside the outer layer of the vessel, or “false,” which is a collection of blood leaking out of an artery or vein. Aneurysms commonly occur, but are not limited to, in arteries at the base of the brain or aortic in the main artery carrying blood from the left ventricle of the heart. In particular, with reference to the aorta, aneurysms can occur at different segments of the aorta including, but not limited to, the beginning of the arch, the end of the arch, the apex, between segments 3 and 5, the supra renal segment, the infra renal segment, before bifurcation, and between the renal artery. Symptoms of aneurysms include pain, peripheral embolization, bleeding and additional symptoms identifiable by a skilled person.

In an embodiment, immunization with one or more of the immunogenic molecules herein described reduces the incidence of experiencing aortic aneurysm rupture (e.g. Examples 2 and 11).

In an embodiment, immunization with one or more of the immunogenic molecules herein described reduces the aortic aneurysmal segment formation. In particular, some of those embodiments, reduction of aneurysms can occur at different segments of the aorta including, but not limited to, the beginning of the arch, the end of the arch, the apex, between segments 3 and 5, the supra renal segment, the infra renal segment, before bifurcation, and between the renal arteries (se e.g. Example 3). The expected reduction of aneurysm after immunization is at least about 20%, and in particular about 20-80% when compared to a control measurement.

In an embodiment, immunization with one or more of the immunogenic molecules herein described reduces mortality associated with aortic aneurysmal rupture (see e.g. Examples 4 and 11).

In an embodiment, immunization with one or more of the immunogenic molecules herein described is associated with a reduction in hypertension.

The term “hypertension” as used herein refers to high blood pressure. In particular, hypertension (HTN) or high blood pressure is a chronic medical condition in which the systemic arterial blood pressure is elevated. It is the opposite of hypotension. It is classified as either primary (essential) or secondary. About 90-95% of cases are termed “primary hypertension”, which refers to high blood pressure for which no medical cause can be found. The remaining 5-10% of cases (Secondary hypertension) is caused by other conditions that affect the kidneys, arteries, heart, or endocrine system.

In an embodiment, immunization with one or more of the immunogenic molecules herein described reduces the incidence of blood pressure (e.g. Examples 10 and 11).

The expected reduction of blood pressure after immunization is at least about 10%, when compared to a control measurement and in particular from about 10% to an amount determined by a physician based on the condition and the individual to be immunized (e.g. Examples 10 and 11).

The term “effective amount” as used herein is meant to describe that amount of antigen, e.g. p210, which induces an antigen-specific immune response.

Effective amounts of an immunogenic fragment and of one or more of the immunogenic molecules herein described to treat and/or prevent a condition will depend on the individual wherein the activation is performed and will be identifiable by a skilled person upon reading of the present disclosure For example in an embodiment a desired effect can be achieved in mice with an effective amount of from about 100 μg to less than about 1000 μg immunogenic fragment or immunogenically active portion thereof.

It has now unexpectedly found that treatment of human individuals with concentration similar to the ones used in mice are particularly effective for all the applications wherein one or more immunogenic fragments of ApoB100 (which include any derivative thereof see related WO 02/080954, PCT application Ser. No. ______ entitled “Immunomodulatory Methods and Systems for Treatment and/or Prevention of Aneurysms” filed on Nov. 11, 2011 with docket number P686-PCT, and to PCT application Ser. No. ______ entitled “Immunomodulatory Methods and Systems for Treatment and/or Prevention of Hypertension” filed on Nov. 11, 2011 with attorney docket P694-PCT each of which is herein incorporated by reference in its entirety), or an immunogenic portion thereof, are administered to humans.

In an embodiment, an effective amount for the treatment or prevention can be about 100 μg or more. In particular, treatment with about 100 μg is expected to prevent aneurysm rupture in humans (see e.g. Example 2).

A greater concentration can be used in some embodiments depending on the desired effect as illustrated in the present disclosure. For example, in embodiments wherein treatment of a condition is expected to be performed with an effective amount be 250 μg or more and in particular with about 500 μg. In another example, an effective amount to treat the condition is expected to be 250 μg or 500 μg or higher is also expected to be effective also depending on other factors affecting the pharmacological activity of the molecule in an individual.

According to the same data, treatment or prevention of a condition can be performed in humans with an effective amount of from about 0.1 to about 100 mg immunogenic fragment or immunogenically active portion thereof.

In particular, the effective amount is expected to vary depending on the number and combination of peptides utilized for each particular vaccine, and specific characteristic and conditions of the individual treated (e.g. immune system, diet and general health and additional factors identifiable by a skilled person). More particular, lower or higher amounts within the defined range are expected to be effective in an individual depending on factors such as weight, age, gender of the individual as well as additional factors identifiable by a skilled person.

There are several well established methods for extrapolating a human equivalent dose (HED) from a dose that is effective in animals. One popular approach is to convert dose per body weight and another is to use an allometric conversion which takes into account body surface area. These approaches are most reliable for small molecules for which the typical sigmoidal dose-response relationship exists. Dose-response relationships for immune modulating therapies are atypical and often unpredictable. This situation is further complicated by the differences in the immune systems of animals and of humans (see attached). Furthermore, the dose that will work in humans need not be an “n” times greater multiple of the dose that worked in animals. It could be the same or even less. There are some in silico (EpiVax) and in vitro (Vax Design, Probiogen) tools that have recently become available that can be used to more accurately predict what formulations/doses will and will not work in humans.

The effective amount is also expected to vary depending on the number and combination of peptides utilized for each particular vaccine, and specific characteristic and conditions of the individual treated (e.g. immune system, diet and general health and additional factors identifiable by a skilled person). More particular, lower or higher amounts within the defined range are expected to be effective in an individual depending on factors such as weight, age, gender of the individual as well as additional factors identifiable by a skilled person. In some embodiments, the immunogenic peptides herein described or related immunogenically active portions can be administered in combination with an adjuvant or other carrier suitable to affect and in particular increase immunogenicity of the peptide o active portion thereof. In particular, in some embodiments, the immunogenic peptide or active portion thereof can be conjugated to the adjuvant or carrier according to procedures identifiable to a skilled person. Suitable carriers comprise BSA, and in particular, cationized BSA, aluminum salts such as aluminum phosphate and aluminum hydroxide and additional carriers identifiable by a skilled person.

In some embodiments, immunogenic molecules herein described can be administered in ratios of immunogenic molecule to carrier to aluminum of about: 1:2:35, 1:2:20.6, 1:2:7.7, 1:2:3.3, 1:1:13.8 weight to weight ratios. In particular, in some embodiments, ratios can be provided wherein the number of peptides conjugated to each carrier molecule while minimizing the amount of aluminum (adjuvant). In particular in one embodiment, ratio can be provided that result in a concentration up to 2.7 mg conjugate/mL.

The route of immunization can vary depending on the purposes of immunization described herein. For example, successful prevention and treatment of aneurysms in mice occurred by subcutaneous route of administration of immunization (Examples 2, 3, and 4). The type of immune response triggered is largely determined by the route of immunization. Various routes can be used comprising subcutaneous, parenteral, and systemic among the others. In particular, the mucosal linings of airways and intestines contain lymphatic tissue that, when exposed to antigen, elicits anti-inflammatory, immunosuppressive responses. Distinct immunological features of the respiratory and intestinal mucosa lead to partly different types of protective immunity upon antigen exposure by the nasal or oral route.

In some embodiments the immunogenic molecules herein described can be administered according to a schedule of administration devised in view of the amount of time required by the adaptive immune system of an individual to mount a response to the initial exposure to an immunogen. Typically, the response is expected to plateau at 2-3 weeks after exposure. Subsequent exposures often elicit a more rapid response. In various embodiments, the following schedules and manner of administration can be followed: (1) single administration, (2) two administrations 2-3 weeks apart, (3) three weekly administrations, (4) up to 6 administrations on a 1 every 3 week schedule. The vaccines have been administered by: (1) subcutaneous injection; (2) intraperitoneal injection; (3)nasal installation; (4) subcutaneous infusion.

In particular, an embodiment, administering one or more immunogenic fragment or an immunogenically active portion thereof can be performed subcutaneously or intramuscularly.

In some embodiments, disclosed are pharmaceutical compositions which contain at least one the immunogenic fragments, active portions thereof, herein described as herein described, in combination with one or more compatible and pharmaceutically acceptable vehicles, and in particular with pharmaceutically acceptable diluents or excipients. In those pharmaceutical compositions the immunogenic fragments, active portions thereof, herein described can be administered as an active ingredient for treatment or prevention of a condition in an individual.

The term “excipient” as used herein indicates an inactive substance used as a carrier for the active ingredients of a medication. Suitable excipients for the pharmaceutical compositions herein disclosed include any substance that enhances the ability of the body of an individual to absorb an immunogenic fragment, active portions thereof herein described. Suitable excipients also include any substance that can be used to bulk up formulations with the immunogenic fragments, active portions thereof, herein described to allow for convenient and accurate dosage. In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of the immunogenic fragments, active portions thereof, herein described. Depending on the route of administration, and form of medication, different excipients can be used. Exemplary excipients include but are not limited to antiadherents, binders, coatings disintegrants, fillers, flavors (such as sweeteners) and colors, glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which is issued to dilute or carry an active ingredient of a composition. Suitable diluent include any substance that can decrease the viscosity of a medicinal preparation.

In some embodiments, pharmaceutical composition can include (1) a peptide or other immunogenic molecule herein described administered alone, (2) a peptide or other immunogenic molecule herein described +carrier(s); (3) a peptide or other immunogenic molecule herein described +adjuvant; (4) a peptide or other immunogenic molecule herein described +carrier +adjuvant. In particular, the carriers for each of the exemplary composition (1) to (4) can comprise: (1) cBSA, (2) rHSA, (3) KLH, (4) cholera toxin subunit B, respectively, each of which can be mineral salt-based. Other carriers, known to those skilled in the art, are expected to be suitable as well as will be identified by a skilled person. Examples of those adjuvants comprise adjuvants having Th2 effects, carriers having adjuvant properties, e.g., diphtheria toxoid, and adjuvants able to function as carriers, e.g., oil-water emulsions. In some embodiments, a necessary, and under certain conditions sufficient, component for the pharmaceutical composition is the immunogenic peptides. Additional components of the composition can be selected to modulate the immunological impact of the peptides or other immunogenic molecule herein described as will be understood by a skilled person.

In an embodiment, any of the immunogenic molecules herein described can be used to specifically activate T cell, and in particular CD8(+) T cells which can be activated using one or more immunogenic fragments of ApoB100 or an immunogenically active portion thereof.

The term “T cells” as used herein indicates T lymphocytes belonging to a group of white blood cells known as lymphocytes, and participate in humoral or cell-mediated immunity. T cells can be distinguished from other lymphocyte types, such as B cells and natural killer cells (NK cells) by the presence of special markers on their cell surface such as T cell receptors (TCR). Additional markers identifying T cell include CD1a, CD3, or additional markers possibly associated to a T cell state and/or functionality as will be understood by a skilled person.

The term “CD8(+) T cells” indicates T cells expressing the CD8 glycoprotein at their surface, wherein the CD8 (cluster of differentiation 8) glycoprotein is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Similarly to the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule, but is specific for the class I MHC protein. Exemplary CD8 T cells comprise cytotoxic memory CD8 T cells, regulatory CD8 T cells, cytotoxic effector CD8 T-cells and additional cells identifiable by a skilled person. There are two isoforms of the protein, alpha and beta, each encoded by a different gene. In humans, both genes are located on chromosome 2 in position 2p12.

The term “activated” and activation as used herein indicate the process by which a T cells interacts with an antigen presenting cell which presents a specific antigen for a time and under condition resulting in a T cell having a preassigned immunological role (e.g. cytotoxicity) within the immune system. The term “antigen-presenting cell” (APC) indicates a cell that displays antigen complex with major histocompatibility complex (MHC) on its surface. T-cells recognize this complex using their T-cell receptor (TCR). Exemplary APCs comprise dendritic cells (DCs) which are known to play an important role in linking innate and acquired immunity, see references (3) (4), and both immune responses participate in atherogenesis, see references (5) (6).

Detection of T cells and in particular, CD8(+) T cells, can be performed by detection of markers such as CD8, alone or in combination with TCR and additional markers identifiable by a skilled person. Detection of activated CD8(+) T cells can be performed by detection of T cells markers and in particular of markers such as CD25, CD44, CD62 and additional markers identifiable by a skilled person using process and techniques suitable for detecting surface markers.

The terms “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a molecule or cell in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and/or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified.

Exemplary techniques suitable for detecting T cell markers comprise use of suitable monoclonal or polyclonal antibodies or antigen-specific HLA or MHC pentamers or hexamers labeled with an appropriate molecule allowing detection as well as additional methods and techniques identifiable by a skilled person. In an exemplary approach T cell markers are identified by flow cytometric analysis as described in the Examples section. Exemplary techniques suitable for detecting T cell markers comprise use of suitable monoclonal or polyclonal antibodies or antigen-specific HLA or MHC pentamers or hexamers labeled with an appropriate molecule allowing detection as well as additional methods and techniques identifiable by a skilled person. In an exemplary approach T cell markers are identified by flow cytometric analysis as described in the Examples section.

In particular, in some embodiments, an effective amount of immunogenic molecules herein described from about 100 μg to less than about 1000 μg is associated with CD8+ Tcell activation that is specific for the activating immunogenic molecule. (see e.g. Example 12).

Additional effective concentrations of immunogenic fragment or immunogenically active portion thereof for specific CD8+ T cell activation comprise concentration from 100 μg to 250 μg and from 250 μg to about 500 μg and from about 0.1 to about 100 mg.

In particular, T cell activation can be performed using any of the molecules herein described administered in vivo in an amount suitable to treat or prevent aneurysms, (see e.g. Example section). Activation of T cell can also be performed in vitro using methods and procedures such as the ones described in ref [52] as well as additional procedures identifiable by a skilled person. Further advantages and characteristics of the present disclosure will become more apparent hereinafter from the following detailed disclosure by way of illustration only with reference to an experimental section.

In some embodiments, activated CD8(+) T cells herein described are expected to be effective in treatment and/or prevention of a condition treatable with the corresponding activating immunogenic molecule herein described. In particular, in some embodiments, activate CD8(+) T cells herein described are expected to be effective according to a schedule of administration wherein those cells are administered to an individual daily (for up to 21 days) and on an every 10 day schedule (days 0, 10, 20). Additional schedules expected to be effective can be identified by a skilled person based on cell treatments of conditions such as HIV and/or cancer.

Further advantages and characteristics of the present disclosure will become more apparent hereinafter from the following detailed disclosure by way of illustration only with reference to an experimental section.

EXAMPLES

The compositions, methods system herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary immunogenic fragments and methods using fragment p210. A person skilled in the art will appreciate the applicability and the necessary modifications to adapt the features described in detail in the present section, to additional immunogenic fragments, administered subcutaneously or using other routes of administration in vivo or in vitro according to embodiments of the present disclosure.

Unless otherwise indicated the following material and methods were followed in the Examples reported below.

Selection of peptides and their preparation for immunization The establishment and screening of human apoB-100 peptides has been reported (8). Based on Applicants pilot experiments and prior reports, see references (9),(10) Applicants selected peptide 210 (p210, KTTKQ SFDLS VKAQY KKNKH—SEQ ID NO: 210) as a candidate immunogen. Native p210 peptide (Euro-Diagnostica AB, Sweden) was conjugated to cationic bovine serum albumin (cBSA) as carrier using a method described previously see references (3), (4) Alum was used as adjuvant and mixed with peptide/cBSA conjugate with 1:1 ratio in volume. Peptide conjugation and mixing with alum were prepared fresh prior to each immunization.

Immunization Protocols

Male apoE (−/−) mice (Jackson Laboratories) were housed in an animal facility accredited by the American Association of Accreditation of Laboratory Animal Care and kept on a 12-hour day/night cycle with unrestricted access to water and food. The Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center approved the experimental protocols. In a pilot experiment, p210 immunization using 100 μg dose conferred optimum athero-reduction compared to 25 or 50 μg dose. Hence 100 μg dose was used for all subsequent experiments. Mice, maintained on normal chow diet, received subcutaneous primary immunization in the dorsal area between scapulas at 6-7 weeks of age, followed by a booster at 9 and 12 weeks of age. One week after last booster, diet was switched to high cholesterol chow (TD 88137, Harlan-Teklad) and continued until euthanasia at the age of 25 weeks. Separate groups of mice receiving PBS or cBSA/alum at the same immunization time-points served as control. Some mice were sacrificed at 8 or 13 weeks of age to assess immune response against p210.

Tissue Harvesting and Preparation

At euthanasia the hearts were harvested and embedded in OCT compound (Tissue-Tek) for cryo-section. Whole aortas were cleaned, processed and stained with Oil Red 0 to assess the extent of atherosclerosis en face with computer-assisted histomorphometry, see references (3),(4).

Immunohistochemistry and Histomorphometry

The sections from aortic sinus were stained with MOMA-2 (Serotec), or CD11c (eBioscience) antibody to identify macrophages or dendritic cells immunohistochemically using standard protocol. Oil-Red-O stain for plaque size was done using standard protocol. Computer-assisted morphometric analysis was performed to assess histomorphometry as described previously, see references (3),(4).

Serum ELISA

Flat-bottomed 96-well polystyrene plates (MaxiSorp, Germany) were pre-coated with 100 ul (20 μg/ml) p210, KLH, TNP-KLH (Biosearch Technologies T-5060) or BSA (2 μg/ml for IgG or 10 μg/ml for IgM) respectively by incubation overnight at 4° C. to assess antibodies levels using standard protocol. The coating concentration was optimized in pilot experiments. Goat anti-mouse HRP-IgG (Pierce 31437) or IgM (Southern Biotech) were used as detecting antibodies and the bound antibodies were detected by developing in ABTS (Southern Biotech) as substrate and optical density values were recorded at 405 nm.

Flow Cytometric Analysis

Flow cytometric analysis was performed using standard protocols with antibodies listed in Table 1 below and a FACScan (Becton Dickinson) or a CyAn ADP analyzer (Beckman Coulter). For intracellular cytokine staining, Brefeldin A (3 μg/ml) was added to the cultured cells for 2 hours before cells subject to staining procedure. Cell membranes were permeabilized for staining intracellular molecules.

TABLE 1 Antigen Clone Type Supplier CD4 GK1.5 FITC-Rat IgG2b, κ BD Pharmingen CD8b.2 53-5.8 FITC-Rat IgG1, κ BD Pharmingen CD25 PC61.5 PE-Rat IgG1, λ eBioscience IL-10 JES5-16E3 Percp-Cy5.5-Rat eBioscience IgG2a, κ IL-12 Clone C17.8 Percp-Cy5.5-Rat eBioscience IgG2b, κ CD11c HL3 FITC-Hamster BD Pharmingen IgG1, λ CD86 GL1 PE-Rat IgG2a, κ BD Pharmingen TGF-Beta 1D11 APC-Mouse IgG1 R&D system Granzyme B 16G6 Alexa-Fluo 647 Rat eBioscience IgG2b, κ Perforin eBioOMAK-D FITC-Rat IgG2a, κ eBioscience

Adoptive Transfer Experiment

Male apoE (−/−) mice on regular chow received subcutaneous immunization as described in previous paragraph and were sacrificed at 13 weeks of age as donors. Splenocytes from the same treatment group were pooled before cell isolation. Donor CD8(+) T-cells, CD4(+)CD25(+) T-cells or B-cells were isolated using Dynabeads FlowComp (Invitrogen) according to the manufacturer's protocols. CD4(+) T-cells were negatively selected from the splenocytes followed by positive selection of CD4(+)CD25(+) cells. B cells were negatively isolated whereas CD8(+) T-cells were positively isolated first and released from beads. The purity of pooled CD8(+) T-cells, CD4(+)CD25(+) T-cells and B-cells was 90%, 80% and 70%, respectively. The isolated CD8(+) T-cells (1×10⁶ cells/mouse), CD4(+)CD25(+) T-cells (1×10⁵ or 3×10⁵ cells/mouse) or B-cells (2×10⁷ cells/mouse) were then adoptively transferred to naïve male apoE (−/−) recipient mice at 6-7 weeks of age via tail vein injection. In the published literatures of vascular biology, the number of adoptively transferred lymphocytes varied greatly. For B-cells transfer, the number of 2×10⁷ cells/mouse was chosen based on two prior reports, see references (11),(12). For CD4(+)CD25(+) T-cells transfer, the number of cells transferred ranged from 5×10⁴ cells/mouse to 1×10⁶ cells/mouse in the published literature see references (13),(14),(15). Hence we chose 2 intermediate doses for our experiment. As to CD8(+) T-cells, 1×10⁶ cells was chosen based on a report from the field of autoimmune disease see ref (16). Applicants did not adoptively transferred CD4(+) T-cells because naïve or antigen-primed CD4(+) T-cells are known to be pro-atherogenic see references (17),(18) Recipient mice were fed normal chow until 13 weeks of age when chow was switched to high cholesterol diet until euthanasia at 25 weeks of age. Aortas were harvested to assess the extent of atherosclerosis.

KLH or Trinitrophenyl-Lipopolysaccharide (TNP-LPS) Immunization

Applicants also tested if p210 immunization affected the efficacy of subsequent immunization with other antigens. KLH was chosen as a prototypical T-cell dependent and TNP as a T-cell independent antigen. Male C57/BL6 mice on regular chow received subcutaneous immunization with p210 conjugate or adjuvant control as described in previous paragraphs for apoE (−/−) mice. At 13 and 15 weeks of age mice were subcutaneously immunized with 100 μg KLH (with alum as adjuvant) at injection sites away from p210 sites or injected intraperitoneally with 100 μg TNP-LPS (Sigma). KLH or TNP immunization was done in separate groups of mice. Blood was collected via retro-orbital puncture at euthanasia (16 weeks of age).

In Vitro Generation of BM-Derived Dendritic Cells (BMDCs)

The method for generating BMDC with GM-CSF was adapted from previous publication with modification see reference (19). Briefly, bone marrow cells from femurs and tibiae of male apoE−/− mice were plated into 10 cm culture plates (Falcon) with 20 ml complete RPMI-1640 containing 10 ng/ml GM-CSF (R&D Systems) and 10 ng/ml IL-4 (Invitrogen). Cells were washed and fed on day 3 and day 5 by removing the old medium followed by replenishing with fresh culture medium with GM-CSF and IL-4. On day 8, the immature DC appeared as non-adherent cells under the microscope and harvested by vigorous pipetting and subcultured into new culture plates with 2×10⁵ DCs in 1.5 ml medium.

In Vitro CD8(+) T-Cells Isolation and Co-Culture with Dendritic Cells

Donor mice [male apoE (−/−) mice] for CD8(+) T-cells were immunized with PBS, cBSA/Alum, or cBSA/Alum/P210 according to the schedule described in earlier paragraphs and splenocytes were harvested at 13 weeks of age. CD8(+) T-cells were negatively isolated using a CD8 selection Dynabeads kit (Invitrogen) per manufacturer's protocol. The selected CD8(+) T-cells were then co-culture with DCs in a CD8:DC ratio of 3:1. A series of pilot studies has been performed to determine the optimal CD8:DC ratio for this assay. After co-culture for 4 hours, cells were collected and processed for flow cytometric determination of CD11c and 7-AAD by LSR II flow cytometer (BD Biosciences) and data was analyzed with Summit V4.3 software. Dendritic cell death without CD8(+) T-cells in the co-culture was used as baseline and percentage of specific lysis of cells was calculated using a method described previously, see reference (20).

Statistics

Data are presented as mean+Std. Number of animals in each group is listed in text or description of the figures. Data were analyzed by ANOVA followed by Newman-Keuls multiple group comparison, or by t-test when appropriate. P<0.05 was considered as statistically significant and horizontal bars in each figure indicated statistically significant difference between groups.

Example 1 Immunogenic Fragments of ApoB-100

Specific immunogenic epitopes by focusing on the single protein found in LDL, apolipoprotein B-100 (apo B) were characterized. A peptide library comprised of 302 peptides, 20 amino acid residues in length, covering the complete 4563 amino acid sequence of human apo B was produced. The peptides were produced with a 5 amino acid overlap to cover all sequences at break points. Peptides were numbered 1-302 starting at the N-terminal of apo B as indicated in Table 1 below.

TABLE 1 Apolipoprotein Peptide Sequence B aa SEQ ID NO P1: EEEML ENVSL VCPKD ATRFK aa 1-20 SEQ ID NO: 1 P2: ATRFK HLRKY TYNYE AESSS aa 16-35 SEQ ID NO: 2 P3: AESSS GVPGT ADSRS ATRIN aa 31-50 SEQ ID NO: 3 P4: ATRIN CKVEL EVPQL CSFIL aa 46-65 SEQ ID NO: 4 P5: CSFIL KTSQC TLKEV YGFNP aa 61-80 SEQ ID NO: 5 P6: YGFNP EGKAL LKKTK NSEEF aa 76-95 SEQ ID NO: 6 P7: NSEEF AAAMS RYELK LAIPE aa 91-110 SEQ ID NO: 7 P8: LAIPE GKQVF LYPEK DEPTY aa 106-125 SEQ ID NO: 8 P9: DEPTY ILNIK RGIIS ALLVP aa 121-140 SEQ ID NO: 9 P10: ALLVP PETEE AKQVL FLDTV aa 136-155 SEQ ID NO: 10 P11: FLDTV YGNCS THFTV KTRKG aa 151-170 SEQ ID NO: 11 P12: KTRKG NVATE ISTER DLGQC aa 166-185 SEQ ID NO: 12 P13: DLGQC DRFKP IRTGI SPLAL aa 181-200 SEQ ID NO: 13 P14: SPLAL IKGMT RPLST LISSS aa 196-215 SEQ ID NO: 14 P15: LISSS QSCQY TLDAK RKHVA aa 211-230 SEQ ID NO: 15 P16: RKHVA EAICK EQHLF LPFSY aa 226-245 SEQ ID NO: 16 P17: LPFSY NNKYG MVAQV TQTLK aa 241-260 SEQ ID NO: 17 P18: TQTLK LEDTP KINSR FFGEG aa 256-275 SEQ ID NO: 18 P19: FFGEG TKKMG LAFES TKSTS aa 271-290 SEQ ID NO: 19 P20: TKSTS PPKQA EAVLK TLQEL aa 286-305 SEQ ID NO: 20 P21: TLQEL KKLTI SEQNI QRANL aa 301-320 SEQ ID NO: 21 P22: QRANL FNKLV TELRG LSDEA aa 316-335 SEQ ID NO: 22 P23: LSDEA VTSLL PQLIE VSSPI aa 331-350 SEQ ID NO: 23 P24: VSSPI TLQAL VQCGQ PQCST aa 346-365 SEQ ID NO: 24 P25: PQCST HILQW LKRVH ANPLL aa 361-380 SEQ ID NO: 25 P26: ANPLL IDVVT YLVAL IPEPS aa 376-395 SEQ ID NO: 26 P27: IPEPS AQQLR EIFNM ARDQR aa 391-410 SEQ ID NO: 27 P28: ARDQR SRATL YALSH AVNNY aa 406-425 SEQ ID NO: 28 P29: AVNNY HKTNP TGTQE LLDIA aa 421-440 SEQ ID NO: 29 P30: LLDIA NYLME QIQDD CTGDE aa 436-455 SEQ ID NO: 30 P31: CTGDE DYTYL ILRVI GNMGQ aa 451-470 SEQ ID NO: 31 P32: GNMGQ TMEQL TPELK SSILK aa 466-485 SEQ ID NO: 32 P33: SSILK CVQST KPSLM IQKAA aa 481-500 SEQ ID NO: 33 P34: IQKAA IQALR KMEPK DKDQE aa 496-515 SEQ ID NO: 34 P35: DKDQE VLLQT FLDDA SPGDK aa 511-530 SEQ ID NO: 35 P36: SPGDK RLAAY LMLMR SPSQA aa 526-545 SEQ ID NO: 36 P37: SPSQA DINKI VQILP WEQNE aa 541-560 SEQ ID NO: 37 P38: WEQNE QVKNF VASHI ANILN aa 556-575 SEQ ID NO: 38 P39: ANILN SEELD IQDLK KLVKE aa 571-590 SEQ ID NO: 39 P40: KLVKE ALKES QLPTV MDFRK aa 586-605 SEQ ID NO: 40 P41: MDFRK FSRNY QLYKS VSLPS aa 601-620 SEQ ID NO: 41 P42: VSLPS LDPAS AKIEG NLIFD aa 616-635 SEQ ID NO: 42 P43: NLIFD PNNYL PKESM LKTTL aa 631-650 SEQ ID NO: 43 P44: LKTTL TAFGF ASADL IEIGL aa 646-665 SEQ ID NO: 44 P45: IEIGL EGKGF EPTLE ALFGK aa 661-680 SEQ ID NO: 45 P46: ALFGK QGFFP DSVNK ALYWV aa 676-695 SEQ ID NO: 46 P47: ALYWV NGQVP DGVSK VLVDH aa 691-710 SEQ ID NO: 47 P48: VLVDH FGYTK DDKHE QDMVN aa 706-725 SEQ ID NO: 48 P49: QDMVN GIMLS VEKLI KDLKS aa 721-740 SEQ ID NO: 49 P50: KDLKS KEVPE ARAYL RILGE aa 736-755 SEQ ID NO: 50 P51: RILGE ELGFA SLHDL QLLGK aa 751-770 SEQ ID NO: 51 P52: QLLGK LLLMG ARTLQ GIPQM aa 766-785 SEQ ID NO: 52 P53: GIPQM IGEVI RKGSK NDFFL aa 781-800 SEQ ID NO: 53 P54: NDFFL HYIFM ENAFE LPTGA aa 796-815 SEQ ID NO: 54 P55: LPTGA GLQLQ ISSSG VIAPG aa 811-830 SEQ ID NO: 55 P56: VIAPG AKAGV KLEVA NMQAE aa 826-845 SEQ ID NO: 56 P57: NMQAE LVAKP SVSVE FVTNM aa 841-860 SEQ ID NO: 57 P58: FVTNM GIIIP DFARS GVQMN aa 856-875 SEQ ID NO: 58 P59: GVQMN TNFFH ESGLE AHVAL aa 871-890 SEQ ID NO: 59 P60: AHVAL KAGKL KFIIP SPKRP aa 886-905 SEQ ID NO: 60 P61: SPKRP VKLLS GGNTL HLVST aa 901-920 SEQ ID NO: 61 P62: HLVST TKTEV IPPLI ENRQS aa 916-935 SEQ ID NO: 62 P63: ENRQS WSVCK QVFPG LNYCT aa 931-950 SEQ ID NO: 63 P64: LNYCT SGAYS NASST DSASY aa 946-965 SEQ ID NO: 64 P65: DSASY YPLTG DTRLE LELRP aa 961-980 SEQ ID NO: 65 P66: LELRP TGEIE QYSVS ATYEL aa 976-995 SEQ ID NO: 66 P67: ATYEL QREDR ALVDT LKFVT aa 991-1010 SEQ ID NO: 67 P68: LKFVT QAEGA KQTEA TMTFK aa 1006-1025 SEQ ID NO: 68 P69: TMTFK YNRQS MTLSS EVQIP aa 1021-1040 SEQ ID NO: 69 P70: EVQIP DFDVD LGTIL RVNDE aa 1036-1055 SEQ ID NO: 70 P71: RVNDE STEGK TSYRL TLDIQ aa 1051-1070 SEQ ID NO: 71 P72: TLDIQ NKKIT EVALM GHLSC aa 1066-1085 SEQ ID NO: 72 P73: GHLSC DTKEE RKIKG VISIP aa 1081-1100 SEQ ID NO: 73 P74: VISIP RLQAE ARSEI LAHWS aa 1096-1115 SEQ ID NO: 74 P75: LAHWS PAKLL LQMDS SATAY aa 1111-1130 SEQ ID NO: 75 P76: SATAY GSTVS KRVAW HYDEE aa 1126-1145 SEQ ID NO: 76 P77: HYDEE KIEFE WNTGT NVDTK aa 1141-1160 SEQ ID NO: 77 P78: NVDTK KMTSN FPVDL SDYPK aa 1156-1175 SEQ ID NO: 78 P79: SDYPK SLHMY ANRLL DHRVP aa 1171-1190 SEQ ID NO: 79 P80: DHRVP ETDMT FRHVG SKLIV aa 1186-1205 SEQ ID NO: 80 P81: SKLIV AMSSW LQKAS GSLPY aa 1201-1220 SEQ ID NO: 81 P82: GSLPY TQTLQ DHLNS LKEFN aa 1216-1235 SEQ ID NO: 82 P83: LKEFN LQNMG LPDFH IPENL aa 1231-1250 SEQ ID NO: 83 P84: IPENL FLKSD GRVKY TLNKN aa 1246-1260 SEQ ID NO: 84 P85: TLNKN SLKIE IPLPF GGKSS aa 1261-1280 SEQ ID NO: 85 P86: GGKSS RDLKM LETVR TPALH aa 1276-1295 SEQ ID NO: 86 P87: TPALH FKSVG FHLPS REFQV aa 1291-1310 SEQ ID NO: 87 P88: REFQV PTFTI PKLYQ LQVPL aa 1306-1325 SEQ ID NO: 88 P89: LQVPL LGVLD LSTNV YSNLY aa 1321-1340 SEQ ID NO: 89 P90: YSNLY NWSAS YSGGN TSTDH aa 1336-1355 SEQ ID NO: 90 P91: TSTDH FSLRA RYHMK ADSVV aa 1351-1370 SEQ ID NO: 91 P92: ADSVV DLLSY NVQGS GETTY aa 1366-1385 SEQ ID NO: 92 P93: GETTY DHKNT FTLSC DGSLR aa 1381-1400 SEQ ID NO: 93 P94: DGSLR HKFLD SNIKF SHVEK aa 1396-1415 SEQ ID NO: 94 P95: SHVEK LGNNP VSKGL LIFDA aa 1411-1430 SEQ ID NO: 95 P96: LIFDA SSSWG PQMSA SVHLD aa 1426-1445 SEQ ID NO: 96 P97: SVHLD SKKKQ HLFVK EVKID aa 1441-1460 SEQ ID NO: 97 P98: EVKID GQFRV SSFYA KGTYG aa 1456-1475 SEQ ID NO: 98 P99: KGTYG LSCQR DPNTG RLNGE aa 1471-1490 SEQ ID NO: 99 P100: RLNGE SNLRF NSSYL QGTNQ aa 1486-1505 SEQ ID NO: 100 P101: QGTNQ ITGRY EDGTL SLTST aa 1501-1520 SEQ ID NO: 101 P102: SLTST SDLQS GIIKN TASLK aa 1516-1535 SEQ ID NO: 102 P103: TASLK YENYE LTLKS DTNGK aa 1531-1550 SEQ ID NO: 103 P104: DTNGK YKNFA TSNKM DMTFS aa 1546-1565 SEQ ID NO: 104 P105: DMTFS KQNAL LRSEY QADYE aa 1561-1580 SEQ ID NO: 105 P106: QADYE SLRFF SLLSG SLNSH aa 1576-1595 SEQ ID NO: 106 P107: SLNSH GLELN ADILG TDKIN aa 1591-1610 SEQ ID NO: 107 P108: TDKIN SGAHK ATLRI GQDGI aa 1606-1625 SEQ ID NO: 108 P109: GQDGI STSAT TNLKC SLLVL aa 1621-1640 SEQ ID NO: 109 P110: SLLVL ENELN AELGL SGASM aa 1636-1655 SEQ ID NO: 110 P111: SGASM KLTTN GRFRE HNAKF aa 1651-1670 SEQ ID NO: 111 P112: HNAKF SLDGK AALTE LSLGS aa 1666-1685 SEQ ID NO: 112 P113: LSLGS AYQAM ILGVD SKNIF aa 1681-1700 SEQ ID NO: 113 P114: SKNIF NFKVS QEGLK LSNDM aa 1696-1715 SEQ ID NO: 114 P115: LSNDM MGSYA EMKFD HTNSL aa 1711-1730 SEQ ID NO: 115 P116: HTNSL NIAGL SLDFS SKLDN aa 1726-1745 SEQ ID NO: 116 P117: SKLDN IYSSD KFYKQ TVNLQ aa 1741-1760 SEQ ID NO: 117 P118: TVNLQ LQPYS LVTTL NSDLK aa 1756-1775 SEQ ID NO: 118 P119: NSDLK YNALD LTNNG KLRLE aa 1771-1790 SEQ ID NO: 119 P120: KLRLE PLKLH VAGNL KGAYQ aa 1786-1805 SEQ ID NO: 120 P121: KGAYQ NNEIK HIYAI SSAAL aa 1801-1820 SEQ ID NO: 121 P122: SSAAL SASYK ADTVA KVQGV aa 1816-1835 SEQ ID NO: 122 P123: KVQGV EFSHR LNTDI AGLAS aa 1831-1850 SEQ ID NO: 123 P124: AGLAS AIDMS TNYNS DSLHF aa 1846-1865 SEQ ID NO: 124 P125: DSLHF SNVFR SVMAP FTMTI aa 1861-1880 SEQ ID NO: 125 P126: FTMTI DAHTN GNGKL ALWGE aa 1876-1895 SEQ ID NO: 126 P127: ALWGE HTGQL YSKFL LKAEP aa 1891-1910 SEQ ID NO: 127 P128: LKAEP LAFTF SHDYK GSTSH aa 1906-1925 SEQ ID NO: 128 P129: GSTSH HLVSR KSISA ALEHK aa 1921-1940 SEQ ID NO: 129 P130: ALEHK VSALL TPAEQ TGTWK aa 1936-1955 SEQ ID NO: 130 P131: TGTWK LKTQF NNNEY SQDLD aa 1951-1970 SEQ ID NO: 131 P132: SQDLD AYNTK DKIGV ELTGR aa 1966-1985 SEQ ID NO: 132 P133: ELTGR TLADL TLLDS PIKVP aa 1981-2000 SEQ ID NO: 133 P134: PIKVP LLLSE PINII DALEM aa 1996-2015 SEQ ID NO: 134 P135: DALEM RDAVE KPQEF TIVAF aa 2011-2030 SEQ ID NO: 135 P136: TIVAF VKYDK NQDVH SINLP aa 2026-2045 SEQ ID NO: 136 P137: SINLP FFLTL QEYFE RNRQT aa 2041-2060 SEQ ID NO: 137 P138: RNRQT IIVVV ENVQR NLKHI aa 2056-2075 SEQ ID NO: 138 P139: NLKHI NIDQF VRKYR AALGK aa 2071-2090 SEQ ID NO: 139 P140: AALGK LPQQA NDYLN SFNWE aa 2086-2105 SEQ ID NO: 140 P141: SFNWE RQVSH AKEKL TALTK aa 2101-2120 SEQ ID NO: 141 P142: TALTK KYRIT ENDIQ IALDD aa 2116-2135 SEQ ID NO: 142 P143: IALDD AKINF NEKLS QLQTY aa 2131-2150 SEQ ID NO: 143 P144: QLQTY MIQFD QYIKD SYDLH aa 2146-2165 SEQ ID NO: 144 P145: SYDLH DLKIA IANII DEIIE aa 2161-2180 SEQ ID NO: 145 P146: DEIIE KLKSL DEHYH IRVNL aa 2176-2195 SEQ ID NO: 146 P147: IRVNL VKTIH DLHLF IENID aa 2191-2210 SEQ ID NO: 147 P148: IENID FNKSG SSTAS WIQNV aa 2206-2225 SEQ ID NO: 148 P149: WIQNV DTKYQ IRIQI QEKLQ aa 2221-2240 SEQ ID NO: 149 P150: QEKLQ QLKRH IQNID IQHLA aa 2236-2255 SEQ ID NO: 150 P151: IQHLA GKLKQ HIEAI DVRVL aa 2251-2270 SEQ ID NO: 151 P152: DVRVL LDQLG TTISF ERIND aa 2266-2285 SEQ ID NO: 152 P153: ERIND VLEHV KHFVI NLIGD aa 2281-2300 SEQ ID NO: 153 P154: NLIGD FEVAE KINAF RAKVH aa 2296-2315 SEQ ID NO: 154 P155: RAKVH ELIER YEVDQ QIQVL aa 2311-2330 SEQ ID NO: 155 P156: QIQVL MDKLV ELTHQ YKLKE aa 2326-2345 SEQ ID NO: 156 P157: YKLKE TIQKL SNVLQ QVKIK aa 2341-2360 SEQ ID NO: 157 P158: QVKIK DYFEK LVGFI DDAVK aa 2356-2375 SEQ ID NO: 158 P159: DDAVK KLNEL SFKTF IEDVN aa 2371-2390 SEQ ID NO: 159 P160: IEDVN KFLDM LIKKL KSFDY aa 2386-2405 SEQ ID NO: 160 P161: KSFDY HQFVD ETNDK IREVT aa 2401-2420 SEQ ID NO: 161 P162: IREVT QRLNG EIQAL ELPQK aa 2416-2435 SEQ ID NO: 162 P163: ELPQK AEALK LFLEE TKATV aa 2431-2450 SEQ ID NO: 163 P164: TKATV AVYLE SLQDT KITLI aa 2446-2465 SEQ ID NO: 164 P165: KITLI INWLQ EALSS ASLAH aa 2461-2480 SEQ ID NO: 165 P166: ASLAH MKAKF RETLE DTRDR aa 2476-2495 SEQ ID NO: 166 P167: DTRDR MYQMD IQQEL QRYLS aa 2491-2510 SEQ ID NO: 167 P168: QRYLS LVGQV YSTLV TYISD aa 2506-2515 SEQ ID NO: 168 P169: TYISD WWTLA AKNLT DFAEQ aa 2521-2540 SEQ ID NO: 169 P170: DFAEQ YSIQD WAKRM KALVE aa 2536-2555 SEQ ID NO: 170 P171: KALVE QGFTV PEIKT ILGTM aa 2551-2570 SEQ ID NO: 171 P172: ILGTM PAFEV SLQAL QKATF aa 2566-2585 SEQ ID NO: 172 P173: QKATF QTPDF IVPLT DLRIP aa 2581-2600 SEQ ID NO: 173 P174: DLRIP SVQIN FKDLK NIKIP aa 2596-2615 SEQ ID NO: 174 P175: NIKIP SRFST PEFTI LNTFH aa 2611-2630 SEQ ID NO: 175 P176: LNTFH IPSFT IDFVE MKVKI aa 2626-2645 SEQ ID NO: 176 P177: MKVKI IRTID QMQNS ELQWP aa 2641-2660 SEQ ID NO: 177 P178: ELQWP VPDIY LRDLK VEDIP aa 2656-2675 SEQ ID NO: 178 P179: VEDIP LARIT LPDFR LPEIA aa 2671-2690 SEQ ID NO: 179 P180: LPEIA IPEFI IPTLN LNDFQ aa 2686-2705 SEQ ID NO: 180 P181: LNDFQ VPDLH IPEFQ LPHIS aa 2701-2720 SEQ ID NO: 181 P182: LPHIS HTIEV PTFGK LYSIL aa 2716-2735 SEQ ID NO: 182 P183: LYSIL KIQSP LFTLD ANADI aa 2731-2750 SEQ ID NO: 183 P184: ANADI GNGTT SANEA GIAAS aa 2746-2765 SEQ ID NO: 184 P185: GIAAS ITAKG ESKLE VLNFD aa 2761-2780 SEQ ID NO: 185 P186: VLNFD FQANA QLSNP KINPL aa 2776-2795 SEQ ID NO: 186 P187: KINPL ALKES VKFSS KYLRT aa 2791-2810 SEQ ID NO: 187 P188: KYLRT EHGSE MLFFG NAIEG aa 2806-2825 SEQ ID NO: 188 P189: NAIEG KSNTV ASLHT EKNTL aa 2821-2840 SEQ ID NO: 189 P190: EKNTL ELSNG VIVKI NNQLT aa 2836-2855 SEQ ID NO: 190 P191: NNQLT LDSNT KYFHK LNIPK aa 2851-2870 SEQ ID NO: 191 P192: LNIPK LDFSS QADLR NEIKT aa 2866-2885 SEQ ID NO: 192 P193: NEIKT LLKAG HIAWT SSGKG aa 2881-2900 SEQ ID NO: 193 P194: SSGKG SWKWA CPRFS DEGTH aa 2896-2915 SEQ ID NO: 194 P195: DEGTH ESQIS FTIEG PLTSF aa 2911-2930 SEQ ID NO: 195 P196: PLTSF GLSNK INSKH LRVNQ aa 2926-2945 SEQ ID NO: 196 P197: LRVNQ NLVYE SGSLN FSKLE aa 2941-2960 SEQ ID NO: 197 P198: FSKLE IQSQV DSQHV GHSVL aa 2956-2975 SEQ ID NO: 198 P199: GHSVL TAKGM ALFGE GKAEF aa 2971-2990 SEQ ID NO: 199 P200: GKAEF TGRHD AHLNG KVIGT aa 2986-3005 SEQ ID NO: 200 P201: KVIGT LKNSL FFSAQ PFEIT aa 3001-3020 SEQ ID NO: 201 P202: PFEIT ASTNN EGNLK VRFPL aa 3016-3035 SEQ ID NO: 202 P203: VRFPL RLTGK IDFLN NYALF aa 3031-3050 SEQ ID NO: 203 P204: NYALF LSPSA QQASW QVSAR aa 3046-3065 SEQ ID NO: 204 P205: QVSAR FNQYK YNQNF SAGNN aa 3061-3080 SEQ ID NO: 205 P206: SAGNN ENIME AHVGI NGEAN aa 3076-3095 SEQ ID NO: 206 P207: NGEAN LDFLN IPLTI PEMRL aa 3091-3110 SEQ ID NO: 207 P208: PEMRL PYTII TTPPL KDFSL aa 3106-3125 SEQ ID NO: 208 P209: KDFSL WEKTG LKEFL KTTKQ aa 3121-3140 SEQ ID NO: 209 P210: KTTKQ SFDLS VKAQY KKNKH aa 3136-3155 SEQ ID NO: 210 P211: KKNKH RHSIT NPLAV LCEFI aa 3151-3170 SEQ ID NO: 211 P212: LCEFI SQSIK SFDRH FLKNR aa 3166-3185 SEQ ID NO: 212 P213: FEKNR NNALD FVTKS YNETK aa 3181-3200 SEQ ID NO: 213 P214: YNETK IKFDK YKAEK SHDEL aa 3196-3215 SEQ ID NO: 214 P215: SHDEL PRTFQ IPGYT VPVVN aa 3211-3230 SEQ ID NO: 215 P216: VPVVN VEVSP FTIEM SAFGY aa 3226-3245 SEQ ID NO: 216 P217: SAFGY VFPKA VSMPS FSILG aa 3241-3260 SEQ ID NO: 217 P218: FSILG SDVRV PSYTL ILPSL aa 3256-3275 SEQ ID NO: 218 P219: ILPSL ELPVL HVPRN LKLSL aa 3271-3290 SEQ ID NO: 219 P220: LKLSL PHFKE LCTIS HIFIP aa 3286-3305 SEQ ID NO: 220 P221: HIFIP AMGNI TYDFS FKSSV aa 3301-3320 SEQ ID NO: 221 P222: FKSSV ITLNT NAELF NQSDI aa 3316-3335 SEQ ID NO: 222 P223: NQSDI VAHLL SSSSS VIDAL aa 3331-3350 SEQ ID NO: 223 P224: VIDAL QYKLE GTTRL TRKRG aa 3346-3365 SEQ ID NO: 224 P225: TRKRG LKLAT ALSLS NKFVE aa 3361-3380 SEQ ID NO: 225 P226: NKFVE GSHNS TVSLT TKNME aa 3376-3395 SEQ ID NO: 226 P227: TKNME VSVAK TTKAE IPILR aa 3391-3410 SEQ ID NO: 227 P228: IPILR MNFKQ ELNGN TKSKP aa 3406-3425 SEQ ID NO: 228 P229: TKSKP TVSSS MEFKY DFNSS aa 3421-3440 SEQ ID NO: 229 P230: DFNSS MLYST AKGAV DHKLS aa 3436-3455 SEQ ID NO: 230 P231: DHKLS LESLT SYFSI ESSTK aa 3451-3470 SEQ ID NO: 231 P232: ESSTK GDVKG SVLSR EYSGT aa 3466-3485 SEQ ID NO: 232 P233: EYSGT IASEA NTYLN SKSTR aa 3481-3500 SEQ ID NO: 233 P234: SKSTR SSVKL QGTSK IDDIW aa 3496-3515 SEQ ID NO: 234 P235: IDDIW NLEVK ENFAG EATLQ aa 3511-3530 SEQ ID NO: 235 P236: EATLQ RIYSL WEHST KNHLQ aa 3526-3545 SEQ ID NO: 236 P237: KNHLQ LEGLF FTNGE HTSKA aa 3541-3560 SEQ ID NO: 237 P238: HTSKA TLELS PWQMS ALVQV aa 3556-3575 SEQ ID NO: 238 P239: ALVQV HASQP SSFHD FPDLG aa 3571-3590 SEQ ID NO: 239 P240: FPDLG QEVAL NANTK NQKIR aa 3586-3605 SEQ ID NO: 240 P241: NQKIR WKNEV RIHSG SFQSQ aa 3601-3620 SEQ ID NO: 241 P242: SFQSQ VELSN DQEKA HLDIA aa 3616-3635 SEQ ID NO: 242 P243: HLDIA GSLEG HLRFL KNIIL aa 3631-3650 SEQ ID NO: 243 P244: KNIIL PVYDK SLWDF LKLDV aa 3646-3665 SEQ ID NO: 244 P245: LKLDV TTSIG RRQHL RVSTA aa 3661-3680 SEQ ID NO: 245 P246: RVSTA FVYTK NPNGY SFSIP aa 3676-3695 SEQ ID NO: 246 P247: SFSIP VKVLA DKFIT PGLKL aa 3691-3710 SEQ ID NO: 247 P248: PGLKL NDLNS VLVMP TFHVP aa 3706-3725 SEQ ID NO: 248 P249: TFHVP FTDLQ VPSCK LDFRE aa 3721-3740 SEQ ID NO: 249 P250: LDFRE IQIYK KLRTS SFALN aa 3736-3755 SEQ ID NO: 250 P251: SFALN LPTLP EVKFP EVDVL aa 3751-3770 SEQ ID NO: 251 P252: EVDVL TKYSQ PEDSL IPFFE aa 3766-3785 SEQ ID NO: 252 P253: IPFFE ITVPE SQLTV SQFTL aa 3781-3800 SEQ ID NO: 253 P254: SQFTL PKSVS DGIAA LDLNA aa 3796-3815 SEQ ID NO: 254 P255: LDLNA VANKI ADFEL PTIIV aa 3811-3830 SEQ ID NO: 255 P256: PTIIV PEQTI EIPSI KFSVP aa 3826-3845 SEQ ID NO: 256 P257: KFSVP AGIVI PSFQA LTARF aa 3841-3860 SEQ ID NO: 257 P258: LTARF EVDSP VYNAT WSASL aa 3856-3875 SEQ ID NO: 258 P259: WSASL KNKAD YVETV LDSTC aa 3871-3890 SEQ ID NO: 259 P260: LDSTC SSTVQ FLEYE LNVLG aa 3886-3905 SEQ ID NO: 260 P261: LNVLG THKIE DGTLA SKTKG aa 3901-3920 SEQ ID NO: 261 P262: SKTKG TLAHR DFSAE YEEDG aa 3916-3935 SEQ ID NO: 262 P263: YEEDG KFEGL QEWEG KAHLN aa 3931-3950 SEQ ID NO: 263 P264: KAHLN IKSPA FTDLH LRYQK aa 3946-3965 SEQ ID NO: 264 P265: LRYQK DKKGI STSAA SPAVG aa 3961-3980 SEQ ID NO: 265 P266: SPAVG TVGMD MDEDD DFSKW aa 3976-3995 SEQ ID NO: 266 P267: DFSKW NFYYS PQSSP DKKLT aa 3991-4010 SEQ ID NO: 267 P268: DKKLT IFKTE LRVRE SDEET aa 4006-4025 SEQ ID NO: 268 P269: SDEET QIKVN WEEEA ASGLL aa 4021-4040 SEQ ID NO: 269 P270: ASGLL TSLKD NVPKA TGVLY aa 4036-4055 SEQ ID NO: 270 P271: TGVLY DYVNK YHWEH TGLTL aa 4051-4070 SEQ ID NO: 271 P272: TGLTL REVSS KLRRN LQNNA aa 4066-4085 SEQ ID NO: 272 P273: LQNNA EWVYQ GAIRQ IDDID aa 4081-4100 SEQ ID NO: 273 P274: IDDID VRFQK AASGT TGTYQ aa 4096-4115 SEQ ID NO: 274 P275: TGTYQ EWKDK AQNLY QELLT aa 4111-4130 SEQ ID NO: 275 P276: QELLT QEGQA SFQGL KDNVF aa 4126-4145 SEQ ID NO: 276 P277: KDNVF DGLVR VTQKF HMKVK aa 4141-4160 SEQ ID NO: 277 P278: HMKVK HLIDS LIDFL NFPRF aa 4156-4175 SEQ ID NO: 278 P279: NFPRF QFPGK PGIYT REELC aa 4171-4190 SEQ ID NO: 279 P280: REELC TMFIR EVGTV LSQVY aa 4186-4205 SEQ ID NO: 280 P281: LSQVY SKVHN GSEIL FSYFQ aa 4201-4220 SEQ ID NO: 281 P282: FSYFQ DLVIT LPFEL RKHKL aa 4216-4235 SEQ ID NO: 282 P283: RKHKL IDVIS MYREL LKDLS aa 4231-4250 SEQ ID NO: 283 P284: LKDLS KEAQE VFKAI QSLKT aa 4246-4265 SEQ ID NO: 284 P285: QSLKT TEVLR NLQDL LQFIF aa 4261-4280 SEQ ID NO: 285 P286: LQFIF QLIED NIKQL KEMKF aa 4276-4295 SEQ ID NO: 286 P287: KEMKF TYLIN YIQDE INTIF aa 4291-4310 SEQ ID NO: 287 P288: INTIF NDYIP YVFKL LKENL aa 4306-4325 SEQ ID NO: 288 P289: LKENL CLNLH KFNEF IQNEL aa 4321-4340 SEQ ID NO: 289 P290: IQNEL QEASQ ELQQI HQYIM aa 4336-4355 SEQ ID NO: 290 P291: HQYIM ALREE YFDPS IVGWT aa 4351-4370 SEQ ID NO: 291 P292: IVGWT VKYYE LEEKI VSLIK aa 4366-4385 SEQ ID NO: 292 P293: VSLIK NLLVA LKDFH SEYIV aa 4381-4400 SEQ ID NO: 293 P294: SEYIV SASNF TSQLS SQVEQ aa 4396-4415 SEQ ID NO: 294 P295: SQVEQ FLHRN IQEYL SILTD aa 4411-4430 SEQ ID NO: 295 P296: SILTD PDGKG KEKIA ELSAT aa 4426-4445 SEQ ID NO: 296 P297: ELSAT AQEII KSQAI ATKKI aa 4441-4460 SEQ ID NO: 297 P298: TKKII SDYHQ QFRYK LQDFS aa 4457-4476 SEQ ID NO: 298 P299: LQDFS DQLSD YYEKF IAESK aa 4472-4491 SEQ ID NO: 299 P300: IAESK RLIDL SIQNY HTFLI aa 4487-4506 SEQ ID NO: 300 P301: HTFLI YITEL LKKLQ STTVM aa 4502-4521 SEQ ID NO: 301 P302: STTVM NPYMK LAPGE LTIIL aa 4517-4536 SEQ ID NO: 302

The full length sequence of ApoB100 can be found in various publications such as San-Hwan Chen et al The complete cDNA and amino acid sequence of Human Apolipoprotein B100 Journal of Biological Chemistry 1986 Vol. 261No 28, Issue of October 5, 12918-12921 (see in particular FIG. 1) herein incorporated by reference in its entirety.

Example 2 Immunization with an apoB-100 Immunogenic Fragments Reduced Aortic Aneurysm Rupture

Male apoE KO mice were subcutaneously immunized at 7, 10, and 12 weeks of age with either Group 1: P210/cBSA conjugate using alum as adjuvant (100 μg P210); Group 2: control-100 μg of cBSA/alum (cBSA); Group 3: control PBS (PBS). Fourteen P210, 17 cBSA, 16 PBS, and 8 Saline injected mice were examined.

AngII (1000 ng/Kg/min) was delivered by a subcutaneous osmotic pump implanted at 10 weeks of age for 4 weeks to cause aneurysms in all three groups. Saline was delivered to the control group. Mice were sacrificed at 14 weeks of age of age. The mice were fed normal chow for the duration of experiment.

Aneurysm formation (including rupture) and incidence were investigated. The results are illustrated in Table 3A and Table 3B below.

TABLE 3A Rupture Aneurysm incidence Pump Group Total alive Rupture (include Rupture) (%, total) AngII p210 14 13 1 8 7.1 cBSA 17 14 3 14 17.7 PBS 16 11 5 11 31.3 Saline Saline 8 8 0 0 0

TABLE 3B Number of mice in Aneurysm Survival at Pump Group each group incidence 28 days Ang II P210 42 54.8% 90.5% cBSA 46 84.8% 69.6% PBS 37 81.1% 64.9% Saline Saline 8   0%  100%

As illustrated in the above Tables, P210 immunization reduced rupture incidence. Immunization with apoB-100 related peptide P210 reduced rupture incidence 7.1% from 17.7% with cBSA as a control and 31.3% using PBS as a control.

A possible mechanism of action provided herein for guidance purposes only and not intended to be limiting is that p210 immunization reduces BP; 2. Effect of p210 immunization is mediated by CD8 to a same or comparable extent detected for reduction of atherosclerosis illustrated in the following examples. Accordingly, ability to elicit a T cell response is specific for p210 (antigen specificity) and other apoB-100 peptides are expected to show similar antigen-specific CD8 effect.

Example 3 Immunization with an Immunogenic Fragment of ApoB-100 Reduces Aortic Aneurysmal Segment Formation

Male apoE KO mice were subcutaneously immunized at 7, 10, and 12 weeks of age with either Group 1: P210/cBSA conjugate using alum as adjuvant (100 μg P210); Group 2: control-100 μg of cBSA/alum (cBSA); Group 3: control PBS (PBS). 42 P210, 46 cBSA, 37 PBS, and 8 Saline injected mice were examined.

AngII (1000 ng/Kg/min) was delivered by a subcutaneous osmotic pump implanted at 10 weeks of age for 4 weeks to cause aneurysms in all three groups. Saline was delivered to the control group. Mice were sacrificed at 14 weeks of age of age. The male apoE KO mice were fed normal chow for the duration of experiment.

The measurement of the aorta was taken at 8 segments: 1) beginning of arch, 2) end of arch, 3) apex level, 4) between 3 & 5, 5) supra renal, 6) infra renal, 7) before bifurcation, and 8) between renal arteries (see schematic illustration of FIG. 1).

The average diameters of each segment illustrated in FIG. 1 are reported in Table 4 below, wherein the segmental aneurysms are circled.

TABLE 4

A further elaboration of the data of Table 4, illustrated in Table 5 below suggests that P210 immunization significantly reduces aneurysmal section formation. Whereas the aneurysmal segment/total segment percentage is 29.6% for cBSA controls and 23.4% for PBS controls, P210 immunization reduced the aneurysmal segment/total segment percentage to 14.3%.

TABLE 5 Aneurysmal segment/total segment (%) p210 14.3 cBSA 29.6 PBS 23.4

Example 4 Immunization with an Immunogenic Fragment of ApoB100 Reduces Mortality Associated with Aortic Aneurysmal Rupture

Male apoE KO mice were subcutaneously immunized at 7, 10, and 12 weeks of age with either Group 1: P210/cBSA conjugate using alum as adjuvant (100 μg P210); Group 2: control-100 μg of cBSA/alum (cBSA); Group 3: control PBS (PBS). 42 P210, 46 cBSA, 37 PBS, and 8 Saline injected mice were examined.

AngII (1000 ng/Kg/min) was delivered by a subcutaneous osmotic pump implanted at 10 weeks of age for 4 weeks to cause aneurysms in all three groups. Saline was delivered to the control group. Mice were sacrificed at 14 weeks of age of age. 42 P210, 46 cBSA, 37 PBS, and 8 Saline injected mice were examined. Mice were sacrificed at 14 weeks of age. The male apoE KO mice were fed normal chow for the duration of experiment.

The results are illustrated in the chart of FIG. 2, which shows survival of the mice treated with p210 over the control groups. The survival rate was 90.5% for P210, 69.6% for cBSA, 64.9% for PBS, and 0% for the saline control at 28 days after implantation of an osmotic pump for angiotensin II infusion to elicit aneurysm formation, as shown in the illustration of FIG. 2.

Example 5 Athero-Protective Effects of p210 Immunization

The vaccine preparation consisted of the p210 peptide (Euro-Diagnostica AB, Sweden) conjugated to cationic bovine serum albumin (cBSA) as carrier using a method described previously^(3;4). Alum was used as adjuvant and mixed with peptide/cBSA conjugated with 1:1 ratio in volume. Peptide conjugation was performed on the day of immunization and freshly mixed with alum just prior to each immunization. Mice fed normal chow diet received subcutaneous primary immunization in the dorsal area between scapulas at 6-7 weeks of age, followed by a booster at 10 and 12 weeks of age. One week after the last booster, diet was switched to high cholesterol chow (TD 88137, Harlan-Teklad) and continued until euthanasia at the age of 25 weeks.

Immunization with p210 reduced aortic atherosclerosis by 57% and 50% compared to PBS and cBSA/Alum group, respectively (FIG. 3A) without affecting circulating cholesterol levels or body weight (Table 6).

TABLE 6 Circulating level of cholesterol and body weight of mice from PBS, cBSA/alum and p210/cBSA/alum group PBS cBSA/alum P210/cBSA/alum P value (n = 10) (n = 10) (n = 10) (ANOVA) Cholesterol 1503 ± 485 1395 ± 420 1135 ± 382 0.17 (mg/dl) Body weight 37.9 ± 5.4 34.8 ± 5.4 34.3 ± 6.5 0.33 (gm)

The aortic sinus plaques from p210/cBSA/alum group contained significantly reduced macrophage and DC immuno-reactivity assessed by MOMA-2 and CD11c immuno-staining, respectively (FIG. 3B) with no difference in the atherosclerotic lesions (PBS group 0.40±0.13 mm², n=10; cBSA/alum group 0.42±0.09 mm², n=10; p210/cBSA/alum group 0.40±0.08 mm², n=9).

Example 6 Characterization of p210-Immunization Elicited Immune Responses

Since DCs are the major cell type upstream to both cellular and humoral immune responses, Applicants determined if these cells were affected by the immunization strategy. Cells from the subcutaneous immunization sites were isolated for flow cytometric analysis one week after primary immunization. The PBS group could not be included in this analysis because mice receiving PBS injection did not develop swelling or cell accumulation at the injection site.

There were significantly fewer CD11c(+) and CD11c(+)CD86(+) cells in p210/cBSA/alum group compared to cBSA/alum group at the immunization site (FIGS. 4A and 4B). When flow cytometry was performed on LN cells 1 week after the third immunization, CD11c(+)CD86(+) cells were also significantly reduced compared with cBSA/alum group (FIG. 4C).

Applicants next assessed antibody response to define the humoral immune response against p210. Before immunization all 3 groups of mice had low levels of IgG titers against p210. At euthanasia, the IgG titer against p210 remained low in the PBS group but was significantly increased in cBSA/alum group. Immunization with p210/cBSA/alum resulted in increased p210 IgG titer compared with PBS group but was significantly reduced compared with cBSA/alum group (FIG. 5A). In contrast to p210 IgG response, there was a significant increase in p210 IgM titer in all groups (FIG. 5B), suggesting an endogenous immune response against p210.

The IL-2Rcc (CD25) is a well-defined lymphocyte activation marker. Applicants therefore analyzed the expression of CD25 on CD4(+) or CD8(+) T-cells from superficial cervical and axillary lymph nodes (LN) from mice one week after primary immunization to assess the T-cell immune response. CD8(+)CD25(+) T-cell population in the lymph nodes was significantly higher in p210/cBSA/alum group when compared to that of PBS or cBSA/alum groups (FIG. 6A) whereas CD4(+)CD25(+) T-cells in the lymph nodes (FIG. 6B) did not differ among 3 groups.

There was a significantly larger population of splenic CD8(+)CD25(+)IL-10(+) T-cells in p210/cBSA/alum group when compared to PBS or cBS A/alum groups (FIG. 6C) without difference in splenic CD8(+)CD25(+)IL12(+) T-cells among 3 groups (FIG. 6D). Splenic CD4(+)CD25(+)IL-10(+) T-cell population significantly increased in the cBSA/alum group. However, this increased response was significantly attenuated by the p210/cBSA/alum immunization (FIG. 6E); whereas splenic CD4(+)CD25(+)IL12(+) T-cells did not differ among the three groups (FIG. 6F).

Example 7 Adoptive Transfer of CD8(+) T-Cells from p210 Immunized Mice to Naive Recipients Recapitulates the Athero-Protective Effect of p210 Immunization

Donor apoE(−/−) mice were subjected to the same immunization protocol with the same groupings, namely: PBS, cBSA/alum, or p210/cBSA/alum. Recipient naïve male apoE(−/−) mice were injected with donor cells at 6-7 weeks of age and were fed normal chow until 13 weeks of age when chow was switched to high cholesterol diet until euthanasia at 25 weeks of age.

At euthanasia, the recipient mice injected with CD8(+) T-cells from p210/cBSA/alum group developed significantly less atherosclerotic lesions in aorta compared to the recipient mice injected with CD8(+) T-cells from PBS or cBSA/alum groups, strongly suggesting that the effector T cell induced by the vaccine are CD8⁺ and is mechanistically involved (FIG. 7A).

This reduction of aortic lesions was coupled with decreased splenic CD11c(+) DCs (PBS group: 4.3±1.7%; cBSA/alum group: 3.4±0.3%; p210/cBSA/alum group: 1.5±0.3%; n=5 each group, p<0.05 p210/cBSA/alum group vs. PBS or cBSA/alum group by ANOVA) with no difference in circulating levels of total cholesterol among 3 groups (PBS group: 1083±296 mg/dl; cBSA/alum group: 975±401 mg/dl; p210/cBSA/alum group: 1098±379 mg/dl).

Adoptive transfer of B cells isolated from the spleens of p210 immunized donor mice did not affect atherosclerosis in recipient mice compared to mice receiving B cells from other donors (FIG. 7B) These observations ruled out B cells as mediators of athero-protective effect of p210 immunization.

To rule out CD4(+)CD25(+) T-cells as possible athero-protective mediators induced by sub-cutaneous p210 immunization, Applicants adoptively transferred CD4(+)CD25(+)T-cells at a dose of 1×10⁵ cells/mouse into naïve recipient apoE−/− mice. There was no difference in lesion size among the 3 groups of CD4(+)CD25(+)T-cell recipients. Depletion of CD25⁺ cells from the pool of CD8⁺ T cells abrogated the reduction in atherosclerosis observed in the p210/cBSA/alum recipient mice, further supporting the notion that CD8⁺ CD25⁺ T cells are mechanistically involved in the protective effects of the vaccine against atherosclerosis (FIG. 7C). Transfer of a higher number of CD4(+)CD25(+) T-cells at 3×10⁵ cells/mouse did not reduce lesion sizes in all 3 recipient groups (FIG. 7D).

Example 8 Increased Cytolytic Activity of CD8(+) T Cells from p210 Immunized Mice Against Dendritic Cells In Vitro

Given the observation that p210 immunization reduced DCs in the immunization sites and atherosclerotic plaques and adoptive transfer of CD8(+) T-cells from p210 immunized donors rendered a decrease of splenic DCs in the recipients, Applicants hypothesized that DCs could be a potential target of CD8(+) T-cells.

To test this, Applicants co-cultured bone marrow derived DCs with CD8(+) T-cells from various immunized groups. CD8(+) T-cells from p210 immunized mice significantly increased the percentage of DC death when compared to those from PBS or BSA/alum groups (FIG. 8). This increased cytolytic function of CD8(+) T-cells was associated with increased granzyme B expression but not perforin (FIG. 9).

Example 9 Immunization with p210 does not Affect the Adaptive Immune Response to Other T-Cell Dependent or Independent Antigens

Given the observations that p210 immunization decreased CD11c(+) DCs and reduced adaptive IgG response to p210, Applicants next tested if such modulation of DCs by p210 immunization would alter the host immune response to other antigens.

Applicants first immunized mice with p210 as described in previous sections followed by two separate subcutaneous KLH immunizations or intra-peritoneal injection of TNP-LPS. Using the KLH- or TNP-IgG titer as a surrogate for the efficacy of individual immunization, Applicants found that there was no difference in KLH- or TNP-IgG titers between p210 immunized mice and the titers from mice of PBS or cBSA/alum groups (FIG. 10).

Example 10 ApoB-100 Related Peptide P210 Immunization Reduces Blood Pressure Induced by Angiotensin

Male apoE KO mice were subcutaneously immunized at 7, 10, and 12 weeks of age with 100 μg of either Group 1: P210/cBSA conjugate using alum as adjuvant (P210); Group 2: control-100 μg of cBSA/alum (cBSA); Group 3: control PBS (PBS). 14 P210, 17 cBSA, 16 PBS, and 8 Saline injected mice were examined.

AngII (1000 ng/Kg/min) was delivered by a subcutaneous osmotic pump implanted at 10 weeks of age for 4 weeks to cause an increase in blood pressure in all three groups. Saline was delivered to the control group. Mice were sacrificed at 14 weeks of age of age. The mice were fed normal chow for the duration of the experiment.

FIG. 11 shows an approximate 11% reduction in blood pressure in P210 vaccinated mice 4 weeks after pump implantation with a concomitant approximate 7% change in hearth rate in P210 vaccinated mice 4 weeks after pump implantation (FIG. 12A). FIG. 12B shows the time course of mean blood pressure change throughout the duration of experiments. Mice received treatment (PBS, cBSA/alum or p210/cBSA/alum) at 7, 10 and 12 weeks of age. Angiotensin II infusion via implanted osmotic pump was started at 10 weeks of age. Mice were euthanized at 14 weeks of age. Blood pressure was measured throughout the duration of experiment. Mean blood pressure gradually increased after angiotensin II infusion was started. At 13 weeks of age, mice immunized with p210/cBSA/alum had a significantly lower mean blood pressure when compared to that in the other 2 groups.

According to the above data it is expected that a p210 vaccine can prevent HTN.

A possible mechanism of action provided herein for guidance purposes only and not intended to be limiting is that p210 immunization reduces BP; and that the effect of p210 immunization is mediated by CD8 to a same or comparable extent detected for reduction of atherosclerosis illustrated in the following examples. Accordingly, ability to elicit a T cell response is specific for p210 (antigen specificity) and other apoB-100 peptides are expected to show similar antigen-specific CD8 effect.

A further possible mechanism of action provided herein for guidance purposes only and not intended to be limiting is that p210 action is performed also through modulation of angiotensin expression. Based on published anti-HTN vaccine literature, an anti-angiotensin vaccine can treat HTN. As a consequence, based on anti-angiotensin vaccine, multiple administration can be desired in certain condition and for certain types of individuals.

Example 11 Immunization with an apoB-100 Immunogenic Fragments Reduces Hypertension and Mortality in Angiotensin II-Induced Aortic Aneurysm

ApoE (−/−) mice were immunized with p210/cBSA/Alum (p210; 100 μg) at 7, 10, and 12 weeks of age. Mice receiving PBS or cBSA/Alum (cBSA) served as controls. At 10 weeks of age, mice were subcutaneously implanted with an osmotic pump which released AngII (1 mg/Kg/min), and were euthanized 4 weeks later. The aorta, spleen, and lymph nodes (LN) were harvested. The p210 vaccine significantly reduced mortality due to AA rupture compared to controls (see FIG. 13).

Flow cytometric analysis of dendritic cells (DCs) in LNs and spleen showed intracellular IFN-γ expression was upregulated in the p210 group. Aortic superoxide production measured by in situ dihydroethidine method and aortic AT1 receptor (AT1R) expression measured by Western blot were significantly decreased in p210 group. The p210 vaccine significantly decreased mean arterial BP at 13 weeks of age (see Table 7).

Mortality from AngII induced AA rupture was significantly reduced by the p210 vaccine. This protective effect was associated with upregulation of IFN-γ expression in DCs and decreased arterial BP, AT1R expression, and superoxide production in aorta. The vaccine may be a promising new non-invasive treatment for AA.

TABLE 7 Flow cytometric analysis of intracellular IFN-γ expression of dendritic cells (DCs) p210 cBSA PBS Spleen CD11c⁺CD86⁺IFN-γ⁺ 19.5 ± 1.6*  13.9 ± 1.4  15.3 ± 0.7 DCs (N = 8 each) LN CD11c⁺CD86⁺IFN-γ⁺ DCs 26.7 ± 1.6*  17.7 ± 2.3  18.1 ± 2.4 (N = 6 each) Aortic AT1R (N = 6 each) 1.0 ± 0.2* 3.1 ± 0.6  3.2 ± 0.5 Aortic superoxide production 1.1 ± 0.1* 1.9 ± 0.2  1.6 ± 0.1 (N = 9 each) Mean Arterial Blood Pressure 124 ± 4*  143 ± 6  139 ± 3  (BP) Spleen and LN DC values are percentage ± SEM of CD11c-gated cells. AT1R values are arbitrary densitometric unit ± SEM. Superoxide values are arbitrary fluorescent intensity unit ± SEM. Mean BP values are mmHg ± SEM at 13 weeks of age; number of mice: p210 N = 9; cBSA N = 7; PBS N = 10. *p < 0.05 vs cBSA and PBS control; ANOVA, followed by post-hoc test.

Example 12 Increased Cytolytic Activity of CD8 (+) T Cells from apoB-100 Immunogenic Fragments Immunized Mice is Specific to Lipid-Associated Antigens

Applicants have shown that immunization with apoB-100 related-peptide p210 significantly reduces atherosclerosis and decreases intra-plaque CD11c⁺ dendritic cells (DCs) in apoE−/− mice. Adoptive transfer experiments showed that athero-protection was mediated by CD8⁺ T cells. Because apoB-100 is found on the LDL fraction of serum lipids, Applicants assessed the CD8⁺ T cell cytolytic activity of p210 immunized mice specific to lipid-associated antigens presented by DCs.

ApoE−/− mice were immunized at 7, 9, and 12 weeks of age with p210/cBSA/alum, cBSA/alum, or PBS. One week after the third immunization, mice were euthanized to collect spleen CD8⁺ T cells. Bone-marrow derived DCs were differentiated from naïve apoE−/− mice and used as target cells. A four-hour lytic assay was performed using a CD8-to-DC ratio of 3:1 in culture medium with 10% FBS. The cells were then collected and stained for CD11c to identify DCs and 7-AAD to assess cell lysis using flow cytometry. There was significantly more lytic activity by CD8⁺ T cells from p210/cBSA/alum immunized mice compared to cBSA/alum and PBS (Table). When the assay was performed in media with delipidated FBS, the lytic activity specific to CD8⁺ T cells from p210/cBSA/alum immunized mice was abrogated (Table 8), suggesting that the lipid fraction of FBS in the culture media provided a source of antigen. Loading of DCs with FITC-labeled p210 24 hours prior to the lytic assay demonstrated antigen uptake and specificity of the lytic activity of CD8⁺ T cells from p210/cBSA/alum immunized mice (see Table 8).

These results show that the cytolytic function of CD8⁺ T cells targeting DCs are specific to lipid-associated antigens, specifically the p210 fragment of apoB-100, and this may underly the protective effects of p210 immunization.

TABLE 8 Flow cytometric analysis of cytolytic activity of CD8 (+) T cells. p210/cBSA/alum cBSA/alum PBS Normal medium (N =  3.7 ± 0.6* 2.7 ± 0.6 2.3 ± 0.8 11 each) Delipidated medium (N = 2.3 ± 0.4 2.4 ± 0.8 2.5 ± 0.5 5 each) FITC-p210 loaded (N = 10.4 ± 0.1† 7.3 ± 0.4 7.8 ± 1.2 3 each) All flow cytometric analysis performed on CD11c-gated cells. CD11c-gated FITC⁺ cells only were assessed in FITC-p210 loaded assay. Values are percent lysis relative to basal lysis. *P < 0.001; †P < 0.01 by ANOVA.

Example 13 Antibody Response to the p210 Vaccine

Antibody titers to p210 was low prior to immunization. At euthanasia at 25 weeks of age, there was a significant increase in p210 IgM titer in all groups (FIG. 14), suggesting an endogenous immune response against self-peptide p210. There was a significant increase in p210 IgG titers in both cBSA/alum group and p210/cBSA/alum compared with the PBS group, but titers in the cBSA/alum was surprisingly the higher between the 2 responding groups. The presence of alum as adjuvant in the cBSA/alum group and p210/cBSA/alum groups likely resulted in class switching of the IgM response to IgG, which did not occur in the PBS group.

Example 14 CD4 (+) T cell and CD8 (+) T Cell Response to the p210 Vaccine

T cells from superficial cervical and axillary lymph nodes (LN) from mice one week after primary immunization were collected to assess the T cell immune response. CD4⁺ CD25⁺ T cells in the lymph nodes (Table 1) did not differ among 3 groups. Splenic CD4⁺ CD25⁺IL-10⁺ T cell population significantly increased in the cBSA/alum group. However, this increased response was significantly attenuated by the p210/cBSA/alum immunization (Table 9). Interestingly, splenic CD4⁺ CD62L⁺ T cell (Table 1) population was lower in cBSA/alum group.

One week after primary immunization, the CD8⁺ CD25⁺ T cell population in the lymph nodes was significantly higher in p210/cBSA/alum group when compared to that of PBS or cBSA/alum groups (Table 2). There was a significantly larger population of splenic CD8⁺ CD25⁺IL-10⁺ T cells in p210/cBSA/alum group when compared to PBS or cBSA/alum groups (Table 2). The splenic CD8⁺ CD62L⁺ T cell population was significantly higher in p210/cBSA/alum group when compared to that of PBS or cBSA/alum groups (Table 9). The T cell profile at other time points were not significantly different between groups.

TABLE 9 CD4 (+) and CD8 (+) T cell response to the p210 vaccine PBS cBSA/alum p210/cBSA/alum CD4⁺ T cell response to p210 vaccine. LN CD4+CD25+ 12.9 ± 1.9  12.5 ± 1.4  14.0 ± 2.8  Spl CD4+CD25+IL-10+ 2.3 ± 0.3  4.3 ± 2.1* 1.7 ± 0.6 Spl CD4+CD62L+ 26.7 ± 1.7  21.4 ± 2.7* 29.9 ± 4.8  CD8⁺ T cell response to p210 vaccine. LN CD8+CD25+ 4.4 ± 0.8 4.1 ± 1.0  6.8 ± 3.0* Spl CD8+CD25+IL-10+ 4.9 ± 3.9 6.0 ± 3.2 12.6 ± 3.9* Spl CD8+CD62L+ 18.4 ± 3.4  19.0 ± 5.5  27.6 ± 5.1* P < 0.05 vs. other groups

Example 15 Effector Role of CD8⁺ CD25⁺ T Cells Involves Cytotoxic Function

The vaccine reduced DC presence in the plaques (FIG. 3), and in the spleens of p210/cBSA/alum recipient mice, suggesting that the effector role of CD8⁺ T cells after immunization was manifested in decreasing DCs in the plaque. Applicants therefore assessed the effect of the vaccine on cytotoxic activity of CD8⁺ T cells against syngeneic bone marrow-derived DCs. CD8⁺ T cells from the immunized groups were negatively isolated using a CD8 selection Dynabeads kit (Invitrogen) followed by co-culture with DCs in a CD8:DC ratio of 3:1 in RPMI supplemented with 10% FBS. Cells were collected and processed for flow cytometric determination of CD11c⁺ and 7-AAD 4 hours later.²⁰ Dendritic cell death without CD8⁺ T cells in the co-culture was used as baseline and percentage of specific lysis of cells was calculated using a method described previously.²⁰

CD8⁺ T cells from p210 immunized mice significantly increased the percentage of DC lysis when compared to those from PBS or cBSA/alum groups (FIG. 15, panel A). This increased cytolytic function of CD8⁺ T cells was associated with increased granzyme B expression but not perforin. Depletion of CD25⁺ cells abrogated the increased cytolytic activity specific to the CD8⁺ T cells from p210 immunized mice (FIG. 15, panel B) indicating that CD8⁺ CD25⁺ T cells were the effector population. The increased cytolytic function specific to CD8⁺ T cells from p210 immunized mice was also lost with the use of delipidated serum supplemented medium (FIG. 15, panel C), indicating that the antigen on the target DCs recognized by the CTLs was derived from serum LDL containing apoB-100 in the medium.

Example 16 p210 Peptide is Endocytosed by DCs In Vitro

Peptide loading on BMDCs was defined using p210 labeled with FITC (FITC conjugating kit from Pierce). The presence of FITC fluorescence in the dendritic cells indicated uptake of p210 by dendritic cells (FIG. 16).

Example 17 p210 Peptide is Presented by DCs to CD8+ T Cells

The p210 peptide contains the proteoglycan binding site of the apoB-100 molecule. This peptide is a cell-penetrating peptide capable of efficiently delivering antigens for cross-presentation to cytotoxic CD8⁺ T cells.⁵³ Applicants therefore assessed activation of CD8⁺ CD25⁻ T cells co-cultured with DCs loaded with p210 and matured with LPS. There was significantly increased CD8⁺ CD25⁺ T cells 48 hours after co-culture with p210-loaded DCs treated with LPS compared to untreated, or LPS only treated co-cultures (FIG. 17). The results suggest that the p210 antigen is presented by DCs to CD8⁺ T cells.

Example 18 p210-Loaded DCs are Specifically Targeted by Immune CD8⁺ T Cells

The results shown above in Example 17 support the notion that p210 is presented by DCs to CD8⁺ T cells. It remained unclear if the lytic activity against DCs was specific to the p210 antigen. Applicants therefore repeated the lytic assay using FITC-labeled p210 loaded BMDC as targets. Lytic activity against FITC DCs was significantly increased in CD8⁺ T cells from the p210/cBSA/alum mice (FIG. 18), indicating antigen specific lytic activity.

In summary, in several embodiments herein described are immunostimulatory agents, T cell, compositions, methods and systems for treating and/or preventing various conditions in a individual and in particular in a human individual.

The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the molecules, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence. Further, the sequence listing submitted herewith in the txt file “P700-PCT-2011-11-11-Sequence Listing_ST25” created on Nov. 11, 2011 forms an integral part of the present application and is incorporated herein by reference in its entirety.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed Thus, it should be understood that although the disclosure has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein can be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.

In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

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1. A method to treat and/or prevent a condition treatable with an immunogenic fragment of ApoB-100 or an immunogenically active portion thereof in an human individual, the method comprising administering to the individual an effective amount of at least one immunogenic fragment of ApoB-100 or the immunogenically active portion thereof, wherein the immunogenic fragment comprises one of SEQ ID NOs 1-302, wherein the effective amount is less than 1 mg, and wherein as a result of the administration, the condition treatable with an immunogenic fragment of ApoB-100 is treated and/or prevented.
 2. The method of claim 1, wherein the immunogenic fragment is associated to atherosclerosis reduction.
 3. (canceled)
 4. The method of claim 1, wherein the one or more immunogenic fragments comprises one or more peptides each comprising one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:45, SEQ ID NO:74, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO:105, SEQ ID NO:129, SEQ ID NO:143, SEQ ID NO:148, SEQ ID NO:210, or SEQ ID NO:301.
 5. The method of claim 1, wherein the one or more immunogenic fragments comprises one or more peptides each comprising one of SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO: 45, SEQ ID NO: 74, SEQ ID NO: 102, SEQ ID NO:148, SEQ ID NO:162, or SEQ ID NO:210.
 6. The method of claim 1, wherein the one or more immunogenic fragments comprises a peptide having SEQ ID NO: 143 and a peptide having SEQ ID NO:
 210. 7. The method of claim 1, wherein the one or more immunogenic fragments comprises a peptide having SEQ ID NO: 11, a peptide having SEQ ID NO: 25 and a peptide having SEQ ID NO:
 74. 8. The method of claim 1, wherein the one or more immunogenic fragments comprises a peptide having SEQ ID NO:
 2. 9. The method of claim 1, wherein the one or more immunogenic fragments comprise a peptide having SEQ ID NO:
 45. 10. The method of claim 1, wherein the one or more immunogenic fragments comprise a peptide having SEQ ID NO:
 210. 11. The method of claim 1, wherein the effective amount is about 100 μg to about 1000 μg.
 12. The method of claim 1, wherein the effective amount is about 100 μg to about 500 μg, or about 500 μg to about less than a 1000 μg.
 13. The method of claim 1, wherein the effective amount is about 100 μg to about 250 μg, or at least about 250 μg.
 14. A pharmaceutical composition comprising less than 1 mg of one or more immunogenic fragments of ApoB-100 or an immunogenically active portion thereof and a pharmaceutically acceptable vehicle, wherein the immunogenic fragment comprises SEQ ID NOs:1-302.
 15. The pharmaceutical composition of claim 14 wherein the composition comprises about 100 μg to about 1000 μg of the one or more immunogenic fragments of ApoB-100 or an immunogenically active portion thereof.
 16. The pharmaceutical composition of claim 14 wherein the composition comprises about 1 μg to about 500 μg, or about 500 μg to about 1000 μg of the one or more immunogenic fragments of ApoB-100 or an immunogenically active portion thereof.
 17. The pharmaceutical composition of claim 14 wherein the composition comprises about 100 μg to about 250 μg or at least about 250 μg of the one or more immunogenic fragments of ApoB-100 or an immunogenically active portion thereof.
 18. A pharmaceutical composition comprising less than a 1 mg of one or more immunogenic fragments of ApoB-100 or an immunogenically active portion thereof and a pharmaceutically acceptable vehicle, wherein the immunogenic fragment consists of one of SEQ ID NOs:1-302. 